Mitochondrial localization of muc1

ABSTRACT

The invention provides methods of identifying and making compounds that inhibit the interaction between MUC1 and either or both of HSP70 and HSP90. Also embraced by the invention are in vivo and in vitro methods of inhibiting such an interaction.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.12/064,425, filed Feb. 21, 2008, which is a national phase applicationunder 35 U.S.C. §371 of International Application No. PCT/US2006/032906,filed Aug. 21, 2006, which claims priority to U.S. provisionalapplication Ser. No. 60/710,166, filed Aug. 22, 2005, the entirecontents of which are hereby incorporated by reference. The entire textof each of the above-referenced disclosures are specificallyincorporated herein by reference without disclaimer.

TECHNICAL FIELD

This invention relates to the regulation of cell growth, and moreparticularly to regulation of cancer cell growth.

BACKGROUND

The MUC1 protein is overexpressed by greater than 800,000 of the 1.3million tumors diagnosed in the United States each year.

SUMMARY

The inventors have found that MUC1 binds to the HSP70 and HSP90chaperones and that this binding is important for targeting of MUC1 tothe mitochondria, where it attenuates stress-induced apoptosis. MUC1binds to HSP70 and HSP90 independently, and c-Src is involved inMUC1-HSP90 binding. The invention includes methods for identifyingcompounds useful for inhibiting the interaction between MUC1 and HSP70or HSP90. Such compounds can be useful for directly promoting apoptosisof MUC1-expressing cancer cells, for enhancing the efficacy of genotoxicchemotherapeutic agents against such cancer cells, and as anti-cancerprophylactic agents. Also included in the invention are methods ofinhibiting the interaction between HSP70 or HSP90 and MUC1 in whichcells (e.g., carcinoma cells such as breast carcinoma cells) arecontacted with compounds that inhibit the interaction between MUC1 andHSP70 or HSP90. While the experiments described herein were generallyperformed with human MUC1, MUC1-binders, and cells, it is understoodthat the methods described herein can be performed with correspondingmolecules from any of the mammalian species recited below.

The invention includes methods of identifying compounds that inhibitbinding of MUC1 to HSP70. The methods include: (a) providing a MUC1 testagent; (b) providing a HSP70 test agent that binds to the MUC1 testagent; (c) contacting the MUC1 test agent with the HSP70 test agent inthe presence of a test compound under conditions that permit the bindingof the MUC1 test agent with the HSP70 test agent in absence of the testcompound; and (d) determining whether the test compound inhibits bindingof the MUC1 test agent to the HSP70 test agent. The contacting can becarried out in a cell-free system or it can occur in a cell.

The invention also includes methods of identifying compounds thatinhibit binding of MUC1 to HSP90. The methods include: (a) providing aMUC1 test agent, e.g., a phosphorylated MUC1 test agent; (b) providing aHSP90 test agent that binds to the MUC1 test agent; (c) contacting theMUC1 test agent with the HSP90 test agent in the presence of a testcompound under conditions that permit the binding of the MUC1 test agentwith the HSP70 test agent in absence of the test compound; and (d)determining whether the test compound inhibits binding of the MUC1 testagent to the HSP90 test agent. The contacting can be carried out in acell-free system or it can occur in a cell. The HSP90 test agent isphosphorylated by c-Src. In some embodiments, the contacting isperformed in the presence of c-Src.

Also featured by the invention are methods of generating compounds thatinhibit the interaction between MUC1 and HSP70 of HSP90. The methodsinclude: (a) providing the three-dimensional structure of a moleculecontaining the cytoplasmic domain of MUC1 or HSP70 or HSP90 (e.g., thesubstrate binding domain of HSP70 or HSP90); (b) designing, based on thethree dimensional structure, a compound containing a region thatinhibits the interaction between MUC1 and HSP70 or the interactionbetween MUC1 and HSP90; and (c) producing the compound.

Another embodiment of the invention is a process of manufacturing acompound. The process includes: (a) performing the method described inthe previous paragraph; and (b) after determining that the compoundinhibits the interaction between MUC1 and HSP70 or MUC1 and HSP90,manufacturing the compound.

In another aspect, the invention provides in vivo methods of inhibitingbinding of MUC1 to HSP70 or HSP90 in a cancer cell that expresses MUC1.The methods include: (a) identifying a subject as having a cancer thatexpresses MUC1 or is suspected to express MUC1; and (b) administering tothe subject a compound or, where the compound is a polypeptide, anucleic acid containing a nucleic acid sequence encoding thepolypeptide, the nucleic acid sequence being operably linked to atranscriptional regulatory element (TRE), wherein the compound inhibitsbinding of HSP70 or HSP90 to the cytoplasmic domain of MUC1. Thecompound can be a peptide fragment of (a) MUC1, (b) HSP70, or (c) HSP90.Thus, the compound can be a peptide fragment of the cytoplasmic domainof MUC1. The compound can be a peptide fragment that includes all orpart of amino acids 46-72 of SEQ ID NO:1. It can be or include all orpart of the substrate binding domain of HSP70 or HSP90. Moreover, thecompound can be an antibody, or an antibody fragment, that binds to thecytoplasmic domain of MUC1. Alternatively, the compound can be a smallmolecule, e.g., a small molecule that is or contains a nucleic acidaptamer. The subject can be a human subject. The cancer cell can be,e.g., a breast cancer, lung cancer, colon cancer, pancreatic cancer,renal cancer, stomach cancer, liver cancer, bone cancer, hematologicalcancer, neural tissue cancer, melanoma, ovarian cancer, testicularcancer, prostate cancer, cervical cancer, vaginal cancer, or bladdercancer cell. The TRE can be a DF3 enhancer.

Also embraced by the invention are methods of killing a cancer cell. Themethods can include, before, after, or at the same time as performingthe methods described in the previous paragraph, exposing the subject toone or more genotoxic agents. The genotoxic agents can be, for example,one or more forms of ionizing radiation and/or one or morechemotherapeutic agents. The one or more chemotherapeutic agents can be,for example, cisplatin, carboplatin, procarbazine, mechlorethamine,cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil,bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin,bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin,tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin,methotrexate, or an analog of any of the aforementioned.

“Polypeptide” and “protein” are used interchangeably and mean anypeptide-linked chain of amino acids, regardless of length orpost-translational modification. The MUC1 and MUC1-binder molecules andtest agents used in any of the methods of the invention can contain orbe wild-type proteins or can be variants that have one or more (e.g.,one, two, three, four, five, six, seven, eight, nine, ten, 12, 15, 20,25, 30, 35, 40, or 50) conservative amino acid substitutions.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine, glutamine, serine andthreonine; lysine, histidine and arginine; and phenylalanine andtyrosine. All that is required is that: (i) such variants of MUC1 haveat least 25% (e.g., at least: 30%; 40%; 50%; 60%; 70%; 75%; 80%; 85%;90%; 95%; 97%; 98%; 99%; 99.5%, or 100% or even greater) of the abilityof wild-type MUC1-C to bind to HSP70 or HSP90; and (ii) such variants ofa MUC1-binder have at least 25% (e.g., at least: 30%; 40%; 50%; 60%;70%; 75%; 80%; 85%; 90%; 95%; 97%; 98%; 99%; 99.5%, or 100% or evengreater) of the ability of the relevant wild-type MUC1-binder to bind toMUC1-C.

As used herein, a “MUC1-binder” is HSP70 or HSP90.

As used herein, a “MUC1-binder test agent” contains, or is, (a) thefull-length, wild-type MUC1-binder, (b) a part of the MUC1-binder thatis shorter than the full-length MUC1-binder, or (c) (a) or (b) but withone or more (see above) conservative substitutions. “Parts of aMUC1-binder” include fragments as well deletion variants (terminal aswell internal deletions) of the MUC1-binder. Deletion variants can lackone, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acid segments (of two or more aminoacids) or non-contiguous single amino acids. MUC1-binder test agents caninclude internal or terminal (C or N) irrelevant amino acid sequences(e.g., sequences derived from other proteins or synthetic sequences notcorresponding to any naturally occurring protein). These addedirrelevant sequences will generally be about 1 to 50 (e.g., two, four,eight, ten, 15, 20, 25, 30, 35, 40, or 45) amino acids in length.MUC1-binder test agents other than full-length wild-type MUC1-bindermolecules will have at least 50% (e.g., at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, atleast 99%, at least 99.5%, or 100% or more) of the ability of thefull-length wild-type MUC1-binder to bind to the cytoplasmic domain ofMUC1.

As used herein, a “MUC1 test agent” contains, or is, (a) full-length,wild-type mature MUC1, (b) a part of MUC1 that is shorter thanfull-length, wild-type, mature MUC1, or (c) (a) or (b) but with one ormore (see above) conservative substitutions. “Parts of a MUC1” includefragments (e.g., MUC1-C or the cytoplasmic domain (CD) of MUC1) as wellas deletion variants (terminal as well internal deletions) of MUC1.Deletion variants can lack one, two, three, four, five, six, seven,eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acidsegments (of two or more amino acids) or non-contiguous single aminoacids. MUC1 test agents can include internal or terminal (carboxy oramino) irrelevant amino acid sequences (e.g., sequences derived fromother proteins or synthetic sequences not corresponding to any naturallyoccurring protein). These added irrelevant sequences will generally beabout 1 to 50 (e.g., two, four, eight, ten, 15, 20, 25, 30, 35, 40, or45) amino acids in length. MUC1 test agents other than full-length,wild-type, mature MUC1 will have at least 50% (e.g., at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, at least 99%, at least 99.5%, or 100% or more) of the abilityof the full-length, wild-type, mature MUC1-binder to bind to HSP70 orHSP90. Both MUC1 test agents and parts of a MUC1 can be phosphorylated,e.g., on a tyrosine residue.

As used herein, “operably linked” means incorporated into a geneticconstruct so that expression control sequences effectively controlexpression of a coding sequence of interest.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can also be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention, e.g., inhibitingsurvival of cancer cells, will be apparent from the followingdescription, from the drawings and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a representation of the pOZ-N-MUC1 vector. To construct thisvector, MUC1-C was cloned into the retroviral pOZ-N vector downstream ofthe Flag-HA epitopes.

FIG. 1B is a reproduction of an SDS-PAGE gel of proteins purified fromHeLa cells stably expressing pOZ-N-MUC1 (MUC1) or the empty vector(Control) by immunoprecipitation with anti-Flag and separation of theprecipitated proteins by glycerol gradient centrifugation. Proteins inthe corresponding gradient fractions were analyzed by SDS-PAGE andCoomassie blue staining MUC1-associated proteins with apparent masses of70 and 90 kDa are highlighted with arrows.

FIGS. 1C and 1D are MALDI-TOF-MS spectra of 70 and 90 kDaimmunoprecipitated proteins. FIG. 1C depicts the analysis of the 70 kDaprotein with peptides corresponding to HSP70 shown with an asterisk.FIG. 1D depicts the analysis of the 90 kDa protein with peptidescorresponding to HSP90 shown with an asterisk.

FIGS. 2A and 2B are series of immunoblots of immunoprecipitated lysates.FIG. 2A depicts immunoblots of lysates from HCT116/MUC1 cells subjectedto immunoprecipitation with anti-MUC1-N or a control mouse IgG. Theprecipitates were analyzed by immunoblotting with the indicatedantibodies (anti-HSP70, anti-HSP90, or anti-MUC1-N). FIG. 2B depictsimmunoblots of immunoprecipitated lysates from HCT116 cells stablyexpressing Flag-MUC1-CD subjected to immunoprecipitation withanti-MUC1-N or a control mouse IgG. The precipitates were analyzed byimmunoblotting with the indicated antibodies (anti-HSP70, anti-HSP90, oranti-MUC1-N).

FIG. 2C depicts the amino acid sequence of MUC1-CD (SEQ ID NO:1). Thec-Src phosphorylation site at Y-46 and the θ-catenin binding domain arehighlighted.

FIG. 2D is a series of immunoblots of adsorbates of recombinant HSP70 orHSP 90 incubated with GST, GST-MUC1-CD, GST-MUC1-CD (1-45) andGST-MUC1-CD (46-72) bound to glutathione beads. The adsorbates wereimmunoblotted with the indicated antibodies (HSP70 and HSP90). Inputlanes show total amounts of HSP70 and HSP90 proteins added to thereactions. Loading of the GST proteins was assessed by Coomassie bluestaining.

FIG. 2E is a series of immunoblots of HSP90 binding to MUC1-CD. Theindicated GST-MUC1-CD fusion proteins were incubated with c-Src and ATPfor 20 minutes at 30° C. HSP90 was then added for 1 hour at 4° C. Theadsorbates were analyzed by immunoblotting with anti-HSP90. Loading ofthe GST proteins was assessed by Coomassie blue staining.

FIG. 3A is a series of immunoblots of immunoprecipitated lysates of 293cells transiently transfected with MUC1 or MUC1 (Y46F) in the presenceand absence of c-Src. Anti-MUC1-N and control IgG immunoprecipitateswere immunoblotted with the indicated antibodies.

FIG. 3B is a bar graph depicting the intensities of the signals in FIG.3B as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 3C is a series of immunoblots of HCT116/MUC1 cell lysatesimmunoprecipitated with anti-c-Src. The cells were untreated prior tolysis or treated with 20 ng/ml HRG for 10 minutes or with 10 mM PP2 for1 hour and then HRG.

FIG. 3D is a series of immunoblots of HCT116/MUC1 cell lysatesimmunoprecipitated with anti-MUC1-N. The cells were untreated prior tolysis or treated with 20 ng/ml HRG for 10 minutes or with 10 mM PP2 or 1mM GA for 1 hour and then HRG. Anti-MUC1-N immunoprecipitates wereimmunoblotted with the indicated antibodies.

FIG. 3E is a bar graph depicting the intensities of the signals in FIG.3D as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 3F is a series of immunoblots of anti-HSP90 immunoprecipitates oflysates of HCT116/vector, HCT116/MUC1 and HCT116/MUC1 (Y46F) cells thatwere untreated or treated with HRG for 10 minutes. Anti-HSP90immunoprecipitates were immunoblotted with the indicated antibodies.

FIG. 3G is a bar graph depicting the intensities of the signals in FIG.3F as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 4A is a series of immunoblots of lysates of MCF-7 cellsimmunoprecipitated with anti-MUC1-N. Cells were untreated or treatedwith HRG for 10 minutes. Anti-MUC1-N immunoprecipitates wereimmunoblotted with the indicated antibodies.

FIG. 4B is a bar graph depicting the intensities of the signals in FIG.4A as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 4C is a series of immunoblots of lysates of MCF-7 cellsimmunoprecipitated with anti-HSP-90. Cells were untreated or treatedwith HRG for 10 minutes. Anti-HSP-90 immunoprecipitates wereimmunoblotted with the indicated antibodies.

FIG. 4D is a bar graph depicting the intensities of the signals in FIG.4C as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 4E is a series of immunoblots of lysates of ZR-75-1 cellsimmunoprecipitated with anti-MUC1-N. Cells were untreated or treatedwith HRG for 10 minutes. Anti-MUC1-N immunoprecipitates wereimmunoblotted with the indicated antibodies.

FIG. 4F is a bar graph depicting the intensities of the signals in FIG.4E as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 4G is a series of immunoblots of lysates of ZR-75-1 cellsimmunoprecipitated with anti-HSP-90. Cells were untreated or treatedwith HRG for 10 minutes. Anti-HSP-90 immunoprecipitates wereimmunoblotted with the indicated antibodies.

FIG. 4H is a bar graph depicting the intensities of the signals in FIG.4G as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 5A is a series of immunoblots of whole cell lysates (WCL) and cellmembrane (CM) preparations of HCT116/MUC1 cells immunoprecipitated withanti-MUC1-N. The cells were left untreated or stimulated with HRG for 10minutes.

FIG. 5B is a bar graph depicting the intensities of the signals in FIG.5A as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 5C is a series of immunoblots of cytosolic fractions of HCT116/MUC1cells immunoblotted with anti-MUC1-C and anti-θ-actin. The cells wereleft untreated or stimulated with HRG for 10 minutes. The cytosolicfractions were also immunoblotted with antibodies against the cellmembrane-associated PDGFR, ER-associated BAP31 andmitochondria-associated Tom20 proteins. Whole cell lysate (WCL) wasincluded as a control.

FIG. 5D is a bar graph depicting the intensities of the signals in FIG.5C as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 5E is a series of immunoblots of anti-HSP90 precipitates fromcytosolic fractions immunoblotted with anti-MUC1-C and anti-HSP90. Thecells were left untreated or stimulated with HRG for 10 minutes.

FIG. 5F is a bar graph depicting the intensities of the signals in FIG.5E as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 5G is a series of immunoblots of anti-Flag precipitates fromHCT116/Flag-MUC1-CD cells that were untreated or treated with HRG for 10minutes. Anti-Flag precipitates were immunoblotted with the indicatedantibodies.

FIG. 5H is a bar graph depicting the intensities of the signals in FIG.5G as assessed by densitometric scanning. The fold-increase inMUC1-HSP90 binding is expressed as the mean±SEM of three separateexperiments compared to that obtained with the MUC1 control (assigned avalue of 1). The asterisk denotes p<0.05 as compared to control.

FIG. 6A is a series of immunoblots of purified mitochondria fromHCT116/MUC1 cells that were treated with HRG for 10 minutes or with PP2for 1 hour and then HRG. Purified mitochondria were subjected toimmunoblotting with the indicated antibodies. Whole cell lysate (WCL)was included as a control. The amount of WCL loaded in the lanerepresents 0.06% of the total protein used to purify the mitochondrialfraction.

FIG. 6B is a series of immunoblots of purified mitochondria fromHCT116/MUC1 cells that were treated with HRG for 10 minutes or with GAfor 1 hour and then HRG. Purified mitochondria were subjected toimmunoblotting with the indicated antibodies. Whole cell lysate (WCL)was included as a control. The amount of WCL loaded in the lanerepresents 0.06% of the total protein used to purify the mitochondrialfraction.

FIG. 6C is a series of immunoblots of mitochondrial or whole cellfractions of HCT116/MUC1 cells that were treated with 1 mM GA for theindicated times. Purified mitochondria (left) and whole cell lysates(right) were immunoblotted with the indicated antibodies.

FIG. 7A is a series of immunoblots of purified mitochondria fromHCT116/MUC1 cells that were untreated or treated with 60 mg/ml trypsinfor 15 minutes at 4° C. Mitochondria were also first incubated inhypotonic buffer before exposure to trypsin. Digestion of the proteinswas analyzed by immunoblotting with the indicated antibodies.

FIG. 7B is a series of immunoblots of purified mitochondria fromHCT116/MUC1 cells that were treated with 0.5% or 1.0% digitonin (DIG)for 15 minutes at 4° C. Lysates were immunoblotted with the indicatedantibodies.

FIG. 7C is a series of immunoblots of purified mitochondria fromHCT116/MUC1 cells that were treated with 0.5% digitonin for 1 minute at4° C., diluted with buffer, and then digested with 60 mg/ml trypsin for15 minutes at 4° C. Digestion of the proteins was analyzed byimmunoblotting with the indicated antibodies.

FIG. 8 is a depiction of a proposed pathway for targeting of MUC1 tomitochondria by HRG/ErbB receptor/c-Src signaling and the HSP70/HSP90complex. TOM, translocase of the mitochondrial outer membrane. TIM,translocase of the mitochondrial inner membrane.

DETAILED DESCRIPTION

MUC1 is a mucin-type glycoprotein that is expressed on the apicalborders of normal secretory epithelial cells (Kufe et al., 1984). MUC1forms a heterodimer following synthesis as a single polypeptide andcleavage of the precursor into two subunits in the endoplasmic reticulum(Ligtenberg et al., 1992). The cleavage may be mediated by anautocatalytic process (Levitan et al., 2005). The >250 kDa MUC1N-terminal (MUC1 N-ter, MUC1-N) subunit contains variable numbers of 20amino acid tandem repeats that are imperfect with highly conservedvariations and are modified by O-linked glycans (Gendler et al., 1988;Siddiqui et al., 1988). MUC1-N is tethered to the cell surface bydimerization with the ˜23 kDa C-terminal subunit (MUC1 C-ter, MUC1-C),which includes a 58 amino acid extracellular region, a 28 amino acidtransmembrane domain and a 72 amino acid cytoplasmic domain (CD; SEQ IDNO:1) (Merlo et al., 1989). The human MUC1 sequence is

The human MUC1-C sequence is:

(SEQ ID NO: 5) GSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSY TNPAVAATSANLWith transformation of normal epithelia to carcinomas, MUC1 isaberrantly overexpressed in the cytosol and over the entire cellmembrane (Kufe et al., 1984; Perey et al., 1992). Cellmembrane-associated MUC1 is targeted to endosomes by clathrin-mediatedendocytosis (Kinlough et al., 2004). In addition, MUC1-C, but notMUC1-N, is targeted to the nucleus (Baldus et al., 2004; Huang et al.,2003; Li et al., 2003a; Li et al., 2003b; Li et al., 2003c; Wei et al.,2005; Wen et al., 2003) and mitochondria (Ren et al., 2004).

MUC1 interacts with members of the ErbB receptor family (Li et al.,2001b; Li et al., 2003c; Schroeder et al., 2001) and with the Wnteffector, θ-catenin (Yamamoto et al., 1997). The epidermal growth factorreceptor and c-Src phosphorylate the MUC1 cytoplasmic domain (MUC1-CD)on Y-46 and thereby increase binding of MUC1 and θ-catenin (Li et al.,2001a; Li et al., 2001b). Binding of MUC1 and θ-catenin is alsoregulated by glycogen synthase kinase 3θ and protein kinase CA (Li etal., 1998; Ren et al., 2002). MUC1 colocalizes with θ-catenin in thenucleus (Baldus et al., 2004; Li et al., 2003a; Li et al., 2003c; Wen etal., 2003) and coactivates transcription of Wnt target genes (Huang etal., 2003). Other studies have shown that MUC1 also binds directly top53 and regulates transcription of p53-target genes (Wei et al., 2005).Notably, overexpression of MUC1 is sufficient to induceanchorage-independent growth and tumorigenicity (Huang et al., 2003; Liet al., 2003b; Ren et al., 2002; Schroeder et al., 2004).

Most mitochondrial proteins are encoded in the nucleus and are importedinto mitochondria by translocation complexes in the outer and innermitochondrial membranes. Certain mitochondrial proteins containN-terminal mitochondrial targeting sequences and interact with Tom20 inthe outer mitochondrial membrane (Truscott et al., 2003). Othermitochondrial proteins contain internal targeting sequences and interactwith the Tom70 receptor (Truscott et al., 2003). Recent work showed thatmitochondrial proteins without internal targeting sequences aredelivered to Tom70 by a complex of HSP70 and HSP90 (Young et al., 2003).

The studies described below show, using tandem affinity purification ofMUC1 complexes and MALDI-TOF-MS, that MUC1 forms intracellular complexeswith HSP70 and HSP90. These results were confirmed by showing that MUC1at the cell membrane and in the cytosol coprecipitates with HSP70 andHSP90 and that the MUC1 cytoplasmic tail is sufficient for conferringthe association with HSP70 and HSP90 in cells. Moreover, MUC1-CDinteracted with HSP70 and HSP90 in vitro. These findings indicate thatMUC1 forms complexes with HSP70 and HSP90, and that these chaperonescontribute to mitochondrial targeting of MUC1.

The human HSP70 sequence is:

(SEQ ID NO: 3) 1msvvgidlgf qscyvavara ggietianey sdrctpacis fgpknrsiga aaksqvisna 61kntvqgfkrf hgrafsdpfv eaeksnlayd ivqwptgltg ikvtymeeer nftteqvtam 121llsklketae svlkkpvvdc vvsvpcfytd aerrsvmdat qiaglnclrl mnettavala 181ygiykqdlpr leekprnvvf vdmghsayqv svcafnrgkl kvlatafdtt lggrkfdevl 241vnhfceefgk kykldikski rallrlsqec eklkklmsan asdlplsiec fmndvdvsgt 301mnrgkflemc ndllarvepp lrsvleqtkl kkediyavei vggatripav kekiskffgk 361elsttlnade avtrgcalqc ailspafkvr efsitdvvpy pislrwnspa eegssdcevf 421sknhaapfsk vltfyrkepf tleayysspq dlpypdpaia qfsvqkvtpq sdgssskvkv 481kvrvnvhgif syssaslvev hkseeneepm etdqnakeee kmqvdqeeph veeqqqqtpa 541enkaeseeme tsqagskdkk mdqppqcqeg ksedqycgpa nresaiwqid remlnlyien 601egkmimqdkl ekerndakna veeyvyemrd klsgeyekfv seddrnsftl kledtenwly 661edgedqpkqv yvdklaelkn lgqpikirfq eseerpnylk n

The human HSP90 sequence is:

(SEQ ID NO: 4) 1mpeevhhgee evetfafqae iaqlmsliin tfysnkeifl relisnasda ldkiryeslt 61dpskldsgke lkidiipnpq ertltivdtg igmtkadlin nlgtiaksgt kafmealqag 121adismigqfg vgfysaylva ekvvvirkhn ddeqyawess aggsftvrad hgepigmgtk 181vilhlkedqt eyleerrvke vvkkhsqfig ypitlyleke rekeisddea eeekgekeee 241dkddeekpki edvgsdeedd sgkdkkkktk kikekyidqe elnktkpiwt rnpdditqee 301ygefyksltn dwedhlavkh fsvegqlefr allfiprrap fdlfenkkkk nniklyvrry 361fimdscdeli peylnfirgv vdsedlpini sremlqqski lkvirknivk kclelfsela 421edkenykkfy eafsknlklg ihedstnrrr lsellryhts qsgdemtsls eyvsrmketq 481ksiyyitges keqvansafv ervrkrgfev vymtepidey cvqqlkefdg kslvsvtkeg 541lelpedeeek kkmeeskakf enlcklmkei ldkkvekvti snrlvsspcc ivtstygwta 601nmerimkaqa lrdnstmgym makkhleinp dhpivetlrq kaeadkndka vkdlvvllfe 661tallssgfsl edpqthsnri yrmiklglgi dedevaaeep naavpdeipp legdedasrm 721eevd

MUC1-C and not MUC1-N is targeted to mitochondria, and this targeting isstimulated by HRG (Ren et al., 2004). Moreover, constitutive andHRG-induced targeting of MUC1-C to mitochondria are both attenuated bythe Y46F mutation (Ren et al., 2004). In this context, c-Src isactivated by HRG (Belsches-Jablonski et al., 2001; Vadlamudi et al.,2003), and c-Src phosphorylates MUC1 on Y-46 (Li et al., 2001a). Thepresent results show that the MUC1 cytoplasmic domain (MUC1-CD) binds toHSP70 and that this interaction occurs independently of c-Src or HRGstimulation. By contrast, binding of MUC1 to HSP90 in vitro wasdependent on c-Src. In cells, it was found that MUC1 bindsconstitutively to HSP90, presumably because of basal levels of Y-46phosphorylation, and that c-Src stimulates this interaction. Moreover,HRG stimulation, which activates c-Src, increased the interactionbetween MUC1 and HSP90 by a mechanism that was attenuated by PP2 or byexpressing MUC1 with the Y46F mutation. These results collectivelysupport a model in which MUC1 binds constitutively to HSP70 and that,with HRG-induced activation of c-Src, phosphorylation of MUC1 on Y-46 inturn stimulates the interaction with HSP90 (FIG. 7D). PP2 alsoattenuated HRG-induced targeting of MUC1 to mitochondria, a finding inconcert with the role of c-Src in inducing MUC1 binding to HSP90 fordelivery to the mitochondrial surface.

The in vitro binding data described below indicate that HSP70 binds tothe MUC1 cytoplasmic tail in the same region as θ-catenin. These datasuggested that MUC1 may form exclusive complexes with θ-catenin and withHSP70. In this regard, there was no detectable θ-catenin associated withMUC1 that was delivered to the mitochondrial outer membrane. Thesefindings collectively support a model in which c-Src phosphorylationregulates HSP90 binding to the client MUC1 protein for delivery tomitochondria.

MUC1 is targeted for integration into the mitochondrial outer membrane.HSP70 functions in the folding of newly synthesized proteins (Bukau etal., 2000; Hartl & Hayer-Hartl, 2002) and cooperates with HSP90 intargeting preproteins to the mitochondrial receptor Tom70 (Young et al.,2003). Binding of HSP70 and HSP90 to Tom70 are in turn required forpreprotein import (Young et al., 2003). GA, an inhibitor of theATP-driven HSP90 chaperone cycle (Young & Hartl, 2000), attenuatedHRG-induced binding of MUC1 to HSP90 and targeting of MUC1 tomitochondria, consistent with delivery by a HSP90-dependent mechanism(FIG. 7D). In further support of HSP90-mediated delivery, GA decreasedthe constitutive localization of MUC1 to mitochondria without affectingtotal intracellular MUC1 pools. In previous work, treatment of purifiedmitochondria with trypsin had no effect on MUC1 (Ren et al., 2004).Moreover, analysis of purified mitochondria incubated in alkaline sodiumcarbonate indicated that MUC1 is an integral membrane protein (Ren etal., 2004). In the present studies, mitochondria were treated withhypotonic buffer to disrupt the outer membrane (Ryan et al., 2001).Under these conditions, trypsin had no effect on MUC1, but decreasedTim23, a mitochondrial inner membrane protein that is exposed in theinter membrane space (Moro et al., 1999). Mitochondria were also treatedwith low levels of digitonin to selectively permeabilize the outermembrane. Permeabilization per se had no effect, but when combined withtrypsin was associated with digestion of MUC1. As controls, thisapproach also resulted in digestion of Tim23, but had little effect onTim44 which is buried in the matrix face of the mitochondrial innermembrane (Truscott et al., 2003; Wada & Kanwar, 1998). These findingsthus collectively support integration and embedding of MUC1 into themitochondrial outer membrane (FIG. 8). MUC1 attenuates release ofmitochondrial apoptogenic factors in the response to stress (Ren et al.,2004). However, integration of MUC1 into the mitochondrial outermembrane could interfere with localization of the proapoptotic Bcl-2subfamily members and thereby neutralization of the antiapoptoticBcl-2/Bcl-XL proteins.

In summary, the above results demonstrate that binding of HSP70 andHSP90 results in localization of MUC1-C to the mitochondria.Mitochondrial MUC1 suppresses apoptosis, e.g., stress-induced apoptosis.

Since MUC1 becomes associated with HSP70 and HSP90 via its physicalassociation with these proteins, compounds that ablate, or at leastinhibit, the interaction between MUC1 and HSP70 and/or HSP90 are likelyto be useful for enhancing the intrinsic apoptosis of cancer cells andalso the cancer cell cytocidal effects of genotoxic agents such asionizing radiation and chemotherapeutic drugs. Moreover, such compoundscould also be useful as prophylactic agents in subjects that have anincreased risk (due, for example, to genetic, physiological, orenvironmental factors) of the development of a malignancy.

Methods of Screening for Inhibitory Compounds

The invention provides in vitro methods for identifying compounds (smallmolecules or macromolecules) that inhibit binding of MUC1-binders (HSP70and HSP90) to MUC1.

These methods can be performed using: (a) isolated MUC1 test agents andMUC1-binder test agents; or (b) cells expressing a MUC1 test agent andone or both MUC1-binder test agents.

The term “isolated” as applied to any of the above-listed polypeptidetest agents refers to a polypeptide, or a peptide fragment thereof,which either has no naturally-occurring counterpart or has beenseparated or purified from components which naturally accompany it,e.g., in tissues such as pancreas, liver, spleen, ovary, testis, muscle,joint tissue, neural tissue, gastrointestinal tissue or tumor tissue(e.g., breast cancer or colon cancer tissue), or body fluids such asblood, serum, or urine. Typically, the polypeptide or peptide fragmentis considered “isolated” when it is at least 70%, by dry weight, freefrom the proteins and other naturally-occurring organic molecules withwhich it is naturally associated. Preferably, a preparation of a testagent is at least 80%, more preferably at least 90%, and most preferablyat least 99%, by dry weight, the test agent. Since a polypeptide that ischemically synthesized is, by its nature, separated from the componentsthat naturally accompany it, a synthetic polypeptide test agent is“isolated.”

An isolated polypeptide test agent can be obtained, for example, byextraction from a natural source (e.g., from tissues); by expression ofa recombinant nucleic acid encoding the polypeptide; or by chemicalsynthesis. A polypeptide test agent that is produced in a cellularsystem different from the source from which it naturally originates is“isolated,” because it will necessarily be free of components whichnaturally accompany it. The degree of isolation or purity can bemeasured by any appropriate method, e.g., column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

Prior to testing, any of the test agents can undergo modification, e.g.,phosphorylation or glycosylation, by methods known in the art. Forexample, a MUC1 test agent can be phosphorylated by c-Src.

In methods of screening for compounds that inhibit or enhance binding ofan isolated MUC1 test agent to an isolated MUC1-binder test agent, aMUC1 test agent is contacted with a MUC1-binder test agent in thepresence of one or more concentrations of a test compound and bindingbetween the two test agents in the presence and absence of the testcompound is detected and/or measured. In such assays neither of the testagents need be detectably labeled. For example, by exploiting thephenomenon of surface plasmon resonance, the MUC1 test agent can bebound to a suitable solid substrate and the MUC1-binder test agentexposed to the substrate-bound MUC1 test agent in the presence andabsence of the compound of interest. Binding of the MUC1-binder testagent to the MUC1 test agent on the solid substrate results in a changein the intensity of surface plasmon resonance that can be detectedqualitatively or quantitatively by an appropriate instrument, e.g., aBiacore3 apparatus (Biacore International AB, Rapsgatan, Sweden). Itwill be appreciated that the experiment can be performed in reverse,i.e., with the MUC1-binder test agent bound to the solid substrate andthe MUC1 test agent added to it in the presence of the test compound.

Moreover, assays to test for inhibition or enhancement of binding toMUC1 can involve the use, for example, of: (a) a single MUC1-specific“detection” antibody that is detectably labeled; (b) an unlabeledMUC1-specific antibody and a detectably labeled secondary antibody; or(c) a biotinylated MUC1-specific antibody and detectably labeled avidin.In addition, combinations of these approaches (including “multi-layer”assays) familiar to those in the art can be used to enhance thesensitivity of assays. In these assays, the MUC1-binder test agent canbe immobilized on a solid substrate such as a nylon or nitrocellulosemembrane by, for example, “spotting” an aliquot of a sample containingthe test agent onto a membrane or by blotting onto a membrane anelectrophoretic gel on which the sample or an aliquot of the sample hasbeen subjected to electrophoretic separation. Alternatively, theMUC1-binder test agent can be bound to a plastic substrate (e.g., theplastic bottom of an ELISA (enzyme-linked immunosorbent assay) platewell using methods known in the art. The substrate-bound test agent isthen exposed to the MUC1 test agent in the presence and absence of thetest compound. After incubating the resulting mixture for a period oftime and at temperature optimized for the system of interest, thepresence and/or amount of MUC1 test agent bound to the MUC1-binder teston the solid substrate is then assayed using a detection antibody thatbinds to the MUC1 test agent and, where required, appropriate detectablylabeled secondary antibodies or avidin. It will be appreciated thatinstead of binding the MUC1-binder test agent to the solid substrate,the MUC1 test agent can be bound to it. In this case binding of theMUC1-binder test agent to the substrate-bound MUC1 is tested by obviousadaptations of the method described above for substrate-boundMUC1-binder test agent.

The invention also features “sandwich” assays. In these sandwich assays,instead of immobilizing test agents on solid substrates by the methodsdescribed above, an appropriate test agent can be immobilized on thesolid substrate by, prior to exposing the solid substrate to the testagent, conjugating a “capture” test agent-specific antibody (polyclonalor mAb) to the solid substrate by any of a variety of methods known inthe art. The test agent is then bound to the solid substrate by virtueof its binding to the capture antibody conjugated to the solidsubstrate. The procedure is carried out in essentially the same mannerdescribed above for methods in which the appropriate test agent is boundto the solid substrate by techniques not involving the use of a captureantibody. It is understood that in these sandwich assays, the captureantibody should not bind to the same epitope (or range of epitopes inthe case of a polyclonal antibody) as the detection antibody. Thus, if amAb is used as a capture antibody, the detection antibody can be either:(a) another mAb that binds to an epitope that is either completelyphysically separated from or only partially overlaps with the epitope towhich the capture mAb binds; or (b) a polyclonal antibody that binds toepitopes other than or in addition to that to which the capture mAbbinds. On the other hand, if a polyclonal antibody is used as a captureantibody, the detection antibody can be either (a) a mAb that binds toan epitope that is either completely physically separated from orpartially overlaps with any of the epitopes to which the capturepolyclonal antibody binds; or (b) a polyclonal antibody that binds toepitopes other than or in addition to that to which the capturepolyclonal antibody binds. Assays that involve the use of a capture anda detection antibody include sandwich ELISA assays, sandwich Westernblotting assays, and sandwich immunomagnetic detection assays.

Suitable solid substrates to which the capture antibody can be boundinclude, without limitation, the plastic bottoms and sides of wells ofmicrotiter plates, membranes such as nylon or nitrocellulose membranes,polymeric (e.g., without limitation, agarose, cellulose, orpolyacrylamide) beads or particles.

Methods of detecting and/or for quantifying a detectable label depend onthe nature of the label and are known in the art. Appropriate labelsinclude, without limitation, radionuclides (e.g., ¹²⁵I, ¹³¹I, ³⁵S, ³H,³²P, or ¹⁴C), fluorescent moieties (e.g., fluorescein, rhodamine, orphycoerythrin), luminescent moieties (e.g., Qdot™ nanoparticles suppliedby the Quantum Dot Corporation, Palo Alto, Calif.), compounds thatabsorb light of a defined wavelength, or enzymes (e.g., alkalinephosphatase or horseradish peroxidase). The products of reactionscatalyzed by appropriate enzymes can be, without limitation,fluorescent, luminescent, or radioactive or they may absorb visible orultraviolet light. Examples of detectors include, without limitation,x-ray film, radioactivity counters, scintillation counters,spectrophotometers, colorimeters, fluorometers, luminometers, anddensitometers.

Candidate compounds can also be tested for their ability to inhibit orenhance binding of MUC1 to a MUC1-binder in cells. The cells can eithernaturally express an appropriate MUC1 test agent and/or MUC1-binder testagent of interest or they can recombinantly express either or both testagents. The cells can be normal or malignant and of any histologicaltype, e.g., without limitation, epithelial cells, fibroblasts, lymphoidcells, macrophages/monocytes, granulocytes, keratinocytes, or musclecells. Suitable cell lines include those recited in the examples, e.g.,breast cancer or colon cancer cell lines. The test compound can be addedto the solution (e.g., culture medium) containing the cells or, wherethe compound is a protein, the cells can recombinantly express it. Thecells can optionally also be exposed to a stimulus of interest (e.g., agrowth factor such as EGF) prior to or after exposure of the cells tothe compound. Following incubation of cells expressing the test agentsof interest in the absence or presence (optionally at variousconcentrations), physical association between the test agents can bedetermined microscopically using appropriately labeled antibodiesspecific for both test agents, e.g., by confocal microscopy.Alternatively, the cells can be lysed under non-dissociating conditionsand the lysates tested for the presence of physically associated testagents. Such methods include adaptations of those described usingisolated test agents. For example, an antibody specific for one of thetwo test agents (test agent 1) can be bound to a solid substrate (e.g.,the bottom and sides of the well of a microtiter plate or a nylonmembrane). After washing away unbound antibody, the solid substrate withbound antibody is contacted with the cell lysate. Any test agent 1 inthe lysate, bound or not bound to the second test agent (test agent 2),will bind to the antibody specific for test agent 1 on the solidsubstrate. After washing away unbound lysate components, the presence oftest agent 2 (bound via test agent 1 and the antibody specific for testagent 1 to the solid substrate) is tested for using a detectably labeledantibody (see above) specific for test agent 2. Alternatively, testagent 1 can be immunoprecipitated with an antibody specific for testagent 1 and the immunoprecipitated material can be subjected toelectrophoretic separation (e.g., by polyacrylamide gel electrophoresisperformed under non-dissociating conditions). The electrophoretic gelcan then be blotted onto a membrane (e.g., a nylon or a nitrocellulosemembrane) and any test agent 2 on the membrane detected and/or measuredwith a detectably labeled antibody (see above) specific for test agent 2by any of the above-described methods. It is understood that in theabove-described assays, test agent 1 can be either the MUC1 test agentor the MUC1-binder test agent or vice versa.

Compounds which may be screened using the assay methods described hereinmay be natural or synthetic chemical compounds. Extracts of plants,microbes or other organisms that contain several characterized oruncharacterized components may also be used. Combinatorial libraries(including solid phase synthesis and parallel synthesis methodologies)provide an efficient means for screening a large numbers of differentsubstances.

Methods of Designing and Producing Inhibitory Compounds

The invention also relates to using MUC1 test agents and/or MUC1-bindertest agents to predict or design compounds that can interact with MUC1and/or MUC1-binders and potentially thereby inhibit the ability of MUC1to interact with an appropriate MUC1-binder. One of skill in the artwould know how to use standard molecular modeling or other techniques toidentify small molecules that would bind to “appropriate sites” on MUC1and/or MUC1-binders. One such example is provided in Broughton (1997)Curr. Opin. Chem. Biol. 1:392-398. Typically, an “appropriate site” on aMUC1 or MUC1-binder is a site directly involved in the physicalinteraction between the two molecule types. However, an “appropriatesite” can also be an allosteric site, i.e., a region of the molecule notdirectly involved in a physical interaction with another molecule (andpossibly even remote from such a “physical interaction” site) but towhich binding of a compound results (e.g., by the induction in aconformational change in the molecule) in inhibition of the binding ofthe molecule to another molecule.

By “molecular modeling” is meant quantitative and/or qualitativeanalysis of the structure and function of protein-protein physicalinteraction based on three-dimensional structural information andprotein-protein interaction models. This includes conventionalnumeric-based molecular dynamic and energy minimization models,interactive computer graphic models, modified molecular mechanicsmodels, distance geometry and other structure-based constraint models.Molecular modeling typically is performed using a computer and may befurther optimized using known methods.

Methods of designing compounds that bind specifically (e.g., with highaffinity) to the region of MUC1 that interacts with HSP70 and/or HSP90(i.e., the cytoplasmic domain of MUC1) or the regions of HSP70 and HSP90that bind to MUC1 (i.e., the substrate binding domains) typically arealso computer-based, and involve the use of a computer having a programcapable of generating an atomic model. Computer programs that use X-raycrystallography data are particularly useful for designing suchcompounds. Programs such as RasMol, for example, can be used to generatea three dimensional model of, e.g., the region of MUC1 that interactswith HSP70 or HSP90 or the region of HSP70 or HSP90 that binds to MUC1and/or determine the structures involved in MUC1-HSP70 or MUC1-HSP90binding. Computer programs such as INSIGHT (Accelrys, Burlington,Mass.), GRASP (Anthony Nicholls, Columbia University), Dock (MolecularDesign Institute, University of California at San Francisco), andAuto-Dock (Accelrys) allow for further manipulation and the ability tointroduce new structures.

Compounds can be designed using, for example, computer hardware orsoftware, or a combination of both. However, designing is preferablyimplemented in one or more computer programs executing on one or moreprogrammable computers, each containing a processor and at least oneinput device. The computer(s) preferably also contain(s) a data storagesystem (including volatile and non-volatile memory and/or storageelements) and at least one output device. Program code is applied toinput data to perform the functions described above and generate outputinformation. The output information is applied to one or more outputdevices in a known fashion. The computer can be, for example, a personalcomputer, microcomputer, or work station of conventional design.

Each program is preferably implemented in a high level procedural orobject oriented programming language to communicate with a computersystem. However, the programs can be implemented in assembly or machinelanguage, if desired. In any case, the language can be a compiled orinterpreted language.

Each computer program is preferably stored on a storage media or device(e.g., ROM or magnetic diskette) readable by a general or specialpurpose programmable computer. The computer program serves to configureand operate the computer to perform the procedures described herein whenthe program is read by the computer. The method of the invention canalso be implemented by means of a computer-readable storage medium,configured with a computer program, where the storage medium soconfigured causes a computer to operate in a specific and predefinedmanner to perform the functions described herein.

For example, the computer-requiring steps in a method of designing acompound can involve:

(a) inputting into an input device, e.g., through a keyboard, adiskette, or a tape, data (e.g. atomic coordinates) that define thethree-dimensional (3-D) structure of a first molecule (e.g., MUC1 or apart of MUC1, or MUC1 or a part of MUC1 that is phosphorylated on atyrosine residue) that binds to a second molecule (e.g., a MUC1-binderor a part thereof) or a molecular complex (e.g., MUC1, or a partthereof, bound to a MUC1-binder, or a part thereof), e.g., a region ofMUC1 that interacts with HSP70 or HSP90 (i.e., the cytoplasmic domain ofMUC1), the region of HSP70 or HSP90 that binds to MUC1 (i.e., thesubstrate binding domain), or all or a part (e.g., the cytoplasmicdomain) of MUC1 bound to all or a part of HSP70 or HSP90; and

(b) determining, using a processor, the 3-D structure (e.g., an atomicmodel) of: (i) the site on the first molecule involved in binding to thesecond molecule; or (ii) one or more sites on the molecular componentsof molecular complex of interaction between molecular components of themolecular complex.

From the information obtained in this way, one skilled in the art willbe able to design and make inhibitory compounds (e.g., peptides,non-peptide small molecules, aptamers (e.g., nucleic acid aptamers) withthe appropriate 3-D structure (see “Methods of Making InhibitoryCompounds and Proteins Useful for the Invention” below).

Moreover, if computer-usable 3-D data (e.g., x-ray crystallographicdata) for a candidate compound are available, the followingcomputer-based steps can be performed in conjunction with computer-basedsteps (a) and (b) described above:

(c) inputting into an input device, e.g., through a keyboard, adiskette, or a tape, data (e.g. atomic coordinates) that define thethree-dimensional (3-D) structure of a candidate compound;

(d) determining, using a processor, the 3-D structure (e.g., an atomicmodel) of the candidate compound;

(e) determining, using the processor, whether the candidate compoundbinds to the site on the first molecule or the one or more sites on themolecular components of the molecular complex; and

(f) identifying the candidate compound as compound that inhibits theinteraction between the first and second molecule or between themolecular components of the molecular complex.

The method can involve the additional step of outputting to an outputdevice a model of the 3-D structure of the compound. In addition, the3-D data of candidate compounds can be compared to a computer databaseof, for example, 3-D structures (e.g., of MUC1, the cytoplasmic domainof MUC1, p53, or the regulatory domain of p53) stored in a data storagesystem.

Compounds of the invention also may be interactively designed fromstructural information of the compounds described herein using otherstructure-based design/modeling techniques (see, e.g., Jackson (1997)Seminars in Oncology 24:L164-172; and Jones et al. (1996) J. Med. Chem.39:904-917). Compounds and polypeptides of the invention also can beidentified by, for example, identifying candidate compounds by computermodeling as fitting spatially and preferentially (i.e., with highaffinity) into the appropriate acceptor sites on MUC1, HSP70, or HSP90.

Candidate compounds identified as described above can then be tested instandard cellular or cell-free binding or binding inhibition assaysfamiliar to those skilled in the art. Exemplary assays are describedherein.

A candidate compound whose presence requires at least 2-fold (e.g.,4-fold, 6-fold, 10-fold, 100-fold, 1000-fold, 10,000 fold, or100,000-fold) more of a given MUC1 test agent to achieve a definedarbitrary level of binding to a fixed amount of a MUC1-binder test agentthan is achieved in the absence of the compound can be useful forinhibiting the interaction between MUC1 and the relevant MUC1-binder,and thus can be useful as a cancer therapeutic or prophylactic agent.Alternatively, a candidate compound whose presence requires at least2-fold (e.g., 2-fold, 4-fold, 6-fold, 10-fold, 100-fold, 1000-fold,10,000 fold, or 100,000-fold) more of a given MUC1-binder test agent toachieve a defined arbitrary level of binding to a fixed amount of a MUC1test agent than is achieved in the absence of the compound can be usefulfor inhibiting the interaction between MUC1 and the relevantMUC1-binder, and thus can be useful as a cancer therapeutic orprophylactic agent.

The 3-D structure of biological macromolecules (e.g., proteins, nucleicacids, carbohydrates, and lipids) can be determined from data obtainedby a variety of methodologies. These methodologies, which have beenapplied most effectively to the assessment of the 3-D structure ofproteins, include: (a) x-ray crystallography; (b) nuclear magneticresonance (NMR) spectroscopy; (c) analysis of physical distanceconstraints formed between defined sites on a macromolecule, e.g.,intramolecular chemical crosslinks between residues on a protein (e.g.,International Patent Application No. PCT/US00/14667, the disclosure ofwhich is incorporated herein by reference in its entirety), and (d)molecular modeling methods based on a knowledge of the primary structureof a protein of interest, e.g., homology modeling techniques, threadingalgorithms, or ab initio structure modeling using computer programs suchas MONSSTER (Modeling Of New Structures from Secondary and TertiaryRestraints) (see, e.g., International Application No. PCT/US99/11913,the disclosure of which is incorporated herein by reference in itsentirety). Other molecular modeling techniques may also be employed inaccordance with this invention (e.g., Cohen et al. (1990) J. Med. Chem.33:883-894; Navia et al. (1992) Curr. Opin. Struct. Biol. 2: 202-210,the disclosures of which are incorporated herein by reference in theirentirety). All these methods produce data that are amenable to computeranalysis. Other spectroscopic methods that can also be useful in themethod of the invention, but that do not currently provide atomic levelstructural detail about biomolecules, include circular dichroism andfluorescence and ultraviolet/visible light absorbance spectroscopy. Apreferred method of analysis is x-ray crystallography. Descriptions ofthis procedure and of NMR spectroscopy are provided below.

X-Ray Crystallography

X-ray crystallography is based on the diffraction of x-radiation of acharacteristic wavelength by electron clouds surrounding the atomicnuclei in a crystal of a molecule or molecular complex of interest. Thetechnique uses crystals of purified biological macromolecules ormolecular complexes (but these frequently include solvent components,co-factors, substrates, or other ligands) to determine near atomicresolution of the atoms making up the particular biologicalmacromolecule. A prerequisite for solving 3-D structure by x-raycrystallography is a well-ordered crystal that will diffract x-raysstrongly. The method directs a beam of x-rays onto a regular, repeatingarray of many identical molecules so that the x-rays are diffracted fromthe array in a pattern from which the structure of an individualmolecule can be retrieved. Well-ordered crystals of, for example,globular protein molecules are large, spherical or ellipsoidal objectswith irregular surfaces. The crystals contain large channels between theindividual molecules. These channels, which normally occupy more thanone half the volume of the crystal, are filled with disordered solventmolecules, and the protein molecules are in contact with each other atonly a few small regions. This is one reason why structures of proteinsin crystals are generally the same as those of proteins in solution.

Methods of obtaining the proteins of interest are described below. Theformation of crystals is dependent on a number of different parameters,including pH, temperature, the concentration of the biologicalmacromolecule, the nature of the solvent and precipitant, as well as thepresence of added ions or ligands of the protein. Many routinecrystallization experiments may be needed to screen all these parametersfor the combinations that give a crystal suitable for x-ray diffractionanalysis. Crystallization robots can automate and speed up work ofreproducibly setting up a large number of crystallization experiments(see, e.g., U.S. Pat. No. 5,790,421, the disclosure of which isincorporated herein by reference in its entirety).

Polypeptide crystallization occurs in solutions in which the polypeptideconcentration exceeds its solubility maximum (i.e., the polypeptidesolution is supersaturated). Such solutions may be restored toequilibrium by reducing the polypeptide concentration, preferablythrough precipitation of the polypeptide crystals. Often polypeptidesmay be induced to crystallize from supersaturated solutions by addingagents that alter the polypeptide surface charges or perturb theinteraction between the polypeptide and bulk water to promoteassociations that lead to crystallization.

Crystallizations are generally carried out between 4° C. and 20° C.Substances known as “precipitants” are often used to decrease thesolubility of the polypeptide in a concentrated solution by forming anenergetically unfavorable precipitating depleted layer around thepolypeptide molecules (Weber (1991) Advances in Protein Chemistry41:1-36). In addition to precipitants, other materials are sometimesadded to the polypeptide crystallization solution. These include buffersto adjust the pH of the solution and salts to reduce the solubility ofthe polypeptide. Various precipitants are known in the art and includethe following: ethanol, 3-ethyl-2-4 pentanediol, and many of thepolyglycols, such as polyethylene glycol (PEG). The precipitatingsolutions can include, for example, 13-24% PEG 4000, 5-41% ammoniumsulfate, and 1.0-1.5 M sodium chloride, and a pH ranging from 5.0-7.5.Other additives can include 0.1 M Hepes, 2-4% butanol, 20-100 mM sodiumacetate, 50-70 mM citric acid, 120-130 mM sodium phosphate, 1 mMethylene diamine tetraacetic acid (EDTA), and 1 mM dithiothreitol (DTT).These agents are prepared in buffers and are added dropwise in variouscombinations to the crystallization buffer. Proteins to be crystallizedcan be modified, e.g., by phosphorylation or by using a phosphate mimic(e.g., tungstate, cacodylate, or sulfate).

Commonly used polypeptide crystallization methods include the followingtechniques: batch, hanging drop, seed initiation, and dialysis. In eachof these methods, it is important to promote continued crystallizationafter nucleation by maintaining a supersaturated solution. In the batchmethod, polypeptide is mixed with precipitants to achievesupersaturation, and the vessel is sealed and set aside until crystalsappear. In the dialysis method, polypeptide is retained in a sealeddialysis membrane that is placed into a solution containing precipitant.Equilibration across the membrane increases the polypeptide andprecipitant concentrations, thereby causing the polypeptide to reachsupersaturation levels.

In the preferred hanging drop technique (McPherson (1976) J. Biol.Chem., 251:6300-6306), an initial polypeptide mixture is created byadding a precipitant to a concentrated polypeptide solution. Theconcentrations of the polypeptide and precipitants are such that in thisinitial form, the polypeptide does not crystallize. A small drop of thismixture is placed on a glass slide that is inverted and suspended over areservoir of a second solution. The system is then sealed. Typically,the second solution contains a higher concentration of precipitant orother dehydrating agent. The difference in the precipitantconcentrations causes the protein solution to have a higher vaporpressure than the second solution. Since the system containing the twosolutions is sealed, an equilibrium is established, and water from thepolypeptide mixture transfers to the second solution. This equilibriumincreases the polypeptide and precipitant concentration in thepolypeptide solution. At the critical concentration of polypeptide andprecipitant, a crystal of the polypeptide may form.

Another method of crystallization introduces a nucleation site into aconcentrated polypeptide solution. Generally, a concentrated polypeptidesolution is prepared and a seed crystal of the polypeptide is introducedinto this solution. If the concentrations of the polypeptide and anyprecipitants are correct, the seed crystal will provide a nucleationsite around which a larger crystal forms.

Yet another method of crystallization is an electrocrystallizationmethod in which use is made of the dipole moments of proteinmacromolecules that self-align in the Helmholtz layer adjacent to anelectrode (see, e.g., U.S. Pat. No. 5,597,457, the disclosure of whichis incorporated herein by reference in its entirety).

Some proteins may be recalcitrant to crystallization. However, severaltechniques are available to the skilled artisan to inducecrystallization. For example, the removal of flexible polypeptidesegments at the amino or carboxyl terminal end of the protein mayfacilitate production of crystalline protein samples. Removal of suchsegments can be done using molecular biology techniques or treatment ofthe protein with proteases such as trypsin, chymotrypsin, or subtilisin.

In diffraction experiments, a narrow and parallel beam of x-rays istaken from the x-ray source and directed onto the crystal to producediffracted beams. The incident primary beams cause damage to both themacromolecule and solvent molecules. The crystal is, therefore, cooled(e.g., to between −220° C. and −50° C.) to prolong its lifetime. Theprimary beam must strike the crystal from many directions to produce allpossible diffraction spots, so the crystal is rotated in the beam duringthe experiment. The diffracted spots are recorded on a film or by anelectronic detector. Exposed film has to be digitized and quantified ina scanning device, whereas the electronic detectors feed the signalsthey detect directly into a computer. Electronic area detectorssignificantly reduce the time required to collect and measurediffraction data. Each diffraction beam, which is recorded as a spot onfilm or a detector plate, is defined by three properties: the amplitude,which is measured from the intensity of the spot; the wavelength, whichis set by the x-ray source; and the phase, which is lost in x-rayexperiments. All three properties are needed for all of the diffractedbeams in order to determine the positions of the atoms giving rise tothe diffracted beams. One way of determining the phases is calledMultiple Isomorphous Replacement (MIR), which requires the introductionof exogenous x-ray scatterers (e.g., heavy atoms such metal atoms) intothe unit cell of the crystal. For a more detailed description of MIR,see U.S. Pat. No. 6,093,573 (column 15) the disclosure of which isincorporated herein by reference in its entirety.

Atomic coordinates refer to Cartesian coordinates (x, y, and zpositions) derived from mathematical equations involving Fouriersynthesis of data derived from patterns obtained via diffraction of amonochromatic beam of x-rays by the atoms (scattering centers) ofbiological macromolecule of interest in crystal form. Diffraction dataare used to calculate electron density maps of repeating units in thecrystal (unit cell). Electron density maps are used to establish thepositions (atomic coordinates) of individual atoms within a crystal'sunit cell. The absolute values of atomic coordinates convey spatialrelationships between atoms because the absolute values ascribed toatomic coordinates can be changed by rotational and/or translationalmovement along x, y, and/or z axes, together or separately, whilemaintaining the same relative spatial relationships among atoms. Thus, abiological macromolecule (e.g., a protein) whose set of absolute atomiccoordinate values can be rotationally or translationally adjusted tocoincide with a set of prior determined values from an analysis ofanother sample is considered to have the same atomic coordinates asthose obtained from the other sample.

Further details on x-ray crystallography can be obtained from co-pendingU.S. Application No. 2005/0015232, U.S. Pat. No. 6,093,573 andInternational Application Nos. PCT/US99/18441, PCT/US99/11913, andPCT/US00/03745. The disclosures of all these patent documents areincorporated herein by reference in their entirety.

NMR Spectroscopy

Whereas x-ray crystallography requires single crystals of amacromolecule of interest, NMR measurements are carried out in solutionunder near physiological conditions. However, NMR-derived structures arenot as detailed as crystal-derived structures.

While the use of NMR spectroscopy was until relatively recently limitedto the elucidation of the 3-D structure of relatively small molecules(e.g., proteins of 100-150 amino acid residues), recent advancesincluding isotopic labeling of the molecule of interest and transverserelaxation-optimized spectroscopy (TROSY) have allowed the methodologyto be extended to the analysis of much larger molecules, e.g., proteinswith a molecular weight of 110 kDa (Wider (2000) BioTechniques,29:1278-1294).

NMR uses radio-frequency radiation to examine the environment ofmagnetic atomic nuclei in a homogeneous magnetic field pulsed with aspecific radio frequency. The pulses perturb the nuclear magnetizationof those atoms with nuclei of nonzero spin. Transient time domainsignals are detected as the system returns to equilibrium. Fouriertransformation of the transient signal into a frequency domain yields aone-dimensional NMR spectrum. Peaks in these spectra represent chemicalshifts of the various active nuclei. The chemical shift of an atom isdetermined by its local electronic environment. Two-dimensional NMRexperiments can provide information about the proximity of various atomsin the structure and in three dimensional space. Protein structures canbe determined by performing a number of two- (and sometimes 3- or 4-)dimensional NMR experiments and using the resulting information asconstraints in a series of protein folding simulations.

More information on NMR spectroscopy including detailed descriptions ofhow raw data obtained from an NMR experiment can be used to determinethe 3-D structure of a macromolecule can be found in: Protein NMRSpectroscopy, Principles and Practice, J. Cavanagh et al., AcademicPress, San Diego, 1996; Gronenborn et al. (1990) Anal. Chem. 62(1):2-15;and Wider (2000), supra., the disclosures of all of which areincorporated herein by reference in their entirety

Any available method can be used to construct a 3-D model of a region ofMUC1 and/or a MUC1-binder of interest from the x-ray crystallographicand/or NMR data using a computer as described above. Such a model can beconstructed from analytical data points inputted into the computer by aninput device and by means of a processor using known software packages,e.g., HKL, MOSFILM, XDS, CCP4, SHARP, PHASES, HEAVY, XPLOR, TNT,NMRCOMPASS, NMRPIPE, DIANA, NMRDRAW, FELIX, VNMR, MADIGRAS, QUANTA,BUSTER, SOLVE, O, FRODO, or CHAIN. The model constructed from these datacan be visualized via an output device of a computer, using availablesystems, e.g., Silicon Graphics, Evans and Sutherland, SUN, HewlettPackard, Apple Macintosh, DEC, IBM, or Compaq.

Methods of Making Inhibitory Compounds and Proteins Useful for theInvention

Once the 3-D structure of a protein of interest (MUC1, HSP70, or HSP90),or a binding region-containing fragment thereof, has been establishedusing any of the above methods, a compound that has substantially thesame 3-D structure (or contains a domain that has substantially the samestructure) as the binding region of the protein of interest can beproduced. The compound's structure can be based on the 3-D structure ofbinding site of the parent protein (e.g., MUC1), the 3-D structure ofthe complementary acceptor site of the protein to which the parentprotein binds (e.g., HSP70 or HSP90), or a combination of both. In thiscontext, “has substantially the same 3-D structure” means that thecompound binds with at least the same avidity as the parent protein tothe non-parent partner. The compound can also bind to the non-parentpartner with at least two-fold (at least: three-fold; four-fold;five-fold; six-fold; seven-fold; eight-fold; nine-fold; ten-fold;20-fold; 50-fold; 100-fold; 1,000-fold; 10,000-fold; 100,000-fold;1,000,000-fold; or even higher-fold) greater avidity than the parentprotein. One of skill in the art would know how to test a compound forsuch an ability.

With the above described 3-D structural data on hand and knowing thechemical structure (e.g., amino acid sequence in the case of a protein)of the protein region of interest, those of skill in the art would knowhow to make compounds with the above-described properties. Such methodsinclude chemical synthetic methods and, in the case of proteins,recombinant methods (see above). For example, cysteine residuesappropriately placed in a compound so as to form disulfide bonds can beused to constrain the compound or a domain of the compound in anappropriate 3-D structure. In addition, in a compound that is apolypeptide or includes a domain that is a polypeptide, one of skill inthe art would know what amino acids to include and in what sequence toinclude them in order to generate, for example, α-helices, β structures,or sharp turns or bends in the polypeptide backbone.

Of particular interest as small molecule compounds are nucleic acidaptamers which are relatively short nucleic acid (DNA, RNA or acombination of both) sequences that bind with high avidity to a varietyof proteins and inhibit the binding to such proteins of ligands,receptors, and other molecules. Aptamers are generally about 25-40nucleotides in length and have molecular weights in the range of about18-25 kDa. Aptamers with high specificity and affinity for targets canbe obtained by an in vitro evolutionary process termed SELEX (systemicevolution of ligands by exponential enrichment) (see, for example, Zhanget al. (2004) Arch. Immunol. Ther. Exp. 52:307-315, the disclosure ofwhich is incorporated herein by reference in its entirety). For methodsof enhancing the stability (by using nucleotide analogs, for example)and enhancing in vivo bioavailability (e.g., in vivo persistence in asubject's circulatory system) of nucleic acid aptamers see Zhang et al.(2004) and Brody et al. (2000) Reviews in Molecular Biotechnology74:5-13, the disclosure of which is incorporated herein by reference inits entirety.

While not essential, computer-based methods can be used to design thecompounds of the invention. Appropriate computer programs include: LUDI(Biosym Technologies, Inc., San Diego, Calif.), Aladdin (DaylightChemical Information Systems, Irvine, Calif.); and LEGEND (Nishibata etal. (1985) J. Med. Chem. 36:2921-2928).

The compounds of the invention can include, in addition, to the abovedescribed domains, one or more domains that facilitate purification(e.g., poly-histidine sequences) or domains that serve to direct thecompound to appropriate target cells (e.g., cancer cells), e.g., ligandsor antibodies (including antibody fragments such as Fab, F(ab′)₂, orsingle chain Fv fragments) specific for cell surface components oftarget cells of the immune system, e.g., MUC1, Her2/Neu, or any of avariety of other tumor-associated antigens (TAA). Signal sequences thatfacilitate transport of the compounds across biological membranes (e.g.,cell membranes and/or nuclear membranes) and/direct them to subcellularcompartments can also be linked (e.g., covalently) to the compounds.Signal sequences are described in detail in U.S. Pat. No. 5,827,516, thedisclosure of which is incorporated herein by reference in its entirety.All that is required in such multidomain compounds is that the domaincorresponding to the parent inhibitory compound substantially retainsthe 3-D structure it would have in the absence of the additionaldomains. Conjugation to make such multidomain compounds can be bychemical methods (e.g., Barrios et al. (1992) Eur. J. Immunol.22:1365-1372, the disclosure of which is incorporated herein byreference in its entirety). Where the compound is a peptide, it can beproduced as part of a recombinant protein, such as one thatself-assembles into virus-sized particles (e.g., U.S. Pat. No.4,918,166, the disclosure of which is incorporated herein by referencein its entirety) that display candidate binding peptides on the surface.

Compounds of the invention that are peptides also include thosedescribed above, but modified for in vivo use by the addition, at theamino- and/or carboxyl-terminal ends, of a blocking agent to facilitatesurvival of the relevant polypeptide in vivo. This can be useful inthose situations in which the peptide termini tend to be degraded byproteases prior to cellular uptake. Such blocking agents can include,without limitation, additional related or unrelated peptide sequencesthat can be attached to the amino and/or carboxyl terminal residues ofthe peptide to be administered. This can be done either chemicallyduring the synthesis of the peptide or by recombinant DNA technology bymethods familiar to artisans of average skill.

Alternatively, blocking agents such as pyroglutamic acid or othermolecules known in the art can be attached to the amino and/or carboxylterminal residues, or the amino group at the amino terminus or carboxylgroup at the carboxyl terminus can be replaced with a different moiety.Likewise, the peptide compounds can be covalently or noncovalentlycoupled to pharmaceutically acceptable “carrier” proteins prior toadministration.

Compounds of the invention that are peptides can also include thosedescribed above, but modified by phosphorylation on a tyrosine residue.In place of a phosphate, the modification may utilize a phosphate mimic,such as tungstate, cacodylate, or sulfate.

Also of interest are peptidomimetic compounds that are designed basedupon the amino acid sequences of compounds of the invention that arepeptides. Peptidomimetic compounds are synthetic compounds having athree-dimensional conformation (i.e., a “peptide motif”) that issubstantially the same as the three-dimensional conformation of aselected peptide. The peptide motif provides the peptidomimetic compoundwith the ability to inhibit the interaction between MUC1 and aMUC1-binder. Peptidomimetic compounds can have additionalcharacteristics that enhance their in vivo utility, such as increasedcell permeability and prolonged biological half-life. Thepeptidomimetics typically have a backbone that is partially orcompletely non-peptide, but with side groups that are identical to theside groups of the amino acid residues that occur in the peptide onwhich the peptidomimetic is based. Several types of chemical bonds,e.g., ester, thioester, thioamide, retroamide, reduced carbonyl,dimethylene and ketomethylene bonds, are known in the art to begenerally useful substitutes for peptide bonds in the construction ofprotease-resistant peptidomimetics.

The proteins (MUC1, HSP70, or HSP90) used for designing compounds of theinvention can be purified from natural sources (e.g., from tissues suchas pancreas, liver, lung, breast, skin, spleen, ovary, testis, muscle,joint tissue, neural tissue, gastrointestinal tissue or tumor tissue(e.g., breast cancer or colon cancer tissue), or body fluids such asblood, serum, or urine). Smaller peptides (fewer than 100 amino acidslong) and other non-protein compounds of the invention can beconveniently synthesized by standard chemical means known to those inthe art. In addition, both polypeptides and peptides can be manufacturedby standard in vitro recombinant DNA techniques and in vivo transgenesisusing nucleotide sequences encoding the appropriate polypeptides orpeptides. Methods well-known to those skilled in the art can be used toconstruct expression vectors containing relevant coding sequences andappropriate transcriptional/translational control signals. See, forexample, the techniques described in Sambrook et al., Molecular Cloning:A Laboratory Manual (2nd Ed.) (Cold Spring Harbor Laboratory, N.Y.,1989), and Ausubel et al., Current Protocols in Molecular Biology (GreenPublishing Associates and Wiley Interscience, N.Y., 1989).

For the structural (e.g., x-ray crystallographic and NMR) analysesdescribed above, it is generally required that proteins, or fragmentsthereof, be highly purified. Methods for purifying biologicalmacromolecules (e.g., proteins) are known in the art. The degree ofpurity of proteins can be measured by any appropriate method, e.g.,column chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

MUC1 and MUC1-binders used for the above analyses can be of anymammalian species, e.g., humans, non-human primates (e.g., monkeys,baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs,cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice.

Methods of Inhibiting Binding of MUC1 to a MUC1-Binder in a Cell

The invention features methods of inhibiting binding of MUC1 to aMUC1-binder (HSP70 or HSP90) in a cell, e.g., a cultured cell or anisolated cell. The method involves introducing into the cell a compoundthat inhibits the binding of a MUC1-binder to the MUC1 (e.g., to theMUC1 CD). Prior to introduction of the compound into the cell, the cell(or another cancer cell from the subject from which the cell to betreated was obtained) can optionally be tested for MUC1 expression. Thiscan be done by testing for expression of either MUC1 protein or MUC1mRNA by any of a wide variety of methods known in the art.

The compound can be one identified by the methods described above.Examples of appropriate compounds include the CD of human MUC1 (SEQ IDNO:1), peptide fragments of the CD of MUC1 that bind to MUC1-binders,and fragments of MUC1-binders that bind MUC1. An appropriate fragment ofthe CD of human MUC1 can be one containing or consisting of amino acids46-72 (YEKVSAGNGGSSLSYTNPAVAATSANL; SEQ ID NO:2) of the human MUC1 CD(SEQ ID NO:1). Other useful inhibitory compounds can be molecules thatcontain or consist of all or part of the substrate binding domains ofhuman HSP70 (SEQ ID NO:3) and HSP90 (SEQ ID NO:4).

Peptide inhibitory compounds can contain up to 50 (e.g., one, two,three, four, five, six, seven, eight, nine, ten, 12, 15, 18, 20, 25, 30,35, 40, 45, or 50) MUC1 or MUC1-binder residues or unrelated residues oneither end or on both ends of the MUC1 or MUC1-binder inhibitorysegments.

Any MUC1 or MUC1-binder peptides to be used as inhibitor compounds canoptionally have any phosphorylation-susceptible amino acid residuesphosphorylated, e.g., Tyr46 of SEQ ID NO:1.

Similarly HSP70 and HSP90 peptide fragment compounds will havesubstantially none of the mitochondrial localizing activity of HSP70 andHSP90 on MUC1. Compounds having substantially none of the mitochondriallocalizing activity of HSP70 and HSP90 on MUC1 have less than 20% (e.g.,less than: 10%; 5%; 2%; 1%; 0.5%; 0.2%; 0.1%; 0.01%; 0.001%; or 0.0001%)of the ability of HSP70 and/or HSP90 to localize MUC1 to mitochondria.Methods of designing, making, and testing such compounds for theappropriate binding-inhibitory activity are known to those in the art.

In addition, the inhibitory compounds can be antibodies, orantigen-binding antibody fragments, specific for MUC1, HSP70, or HSP90.Such antibodies will generally bind to, or close to: (a) the region ofMUC1 to which HSP70 or HSP90 binds; (b) or the region on HSP70 or theregion on HSP90 to which MUC1 binds (i.e., the substrate bindingdomain). However, as indicated above, the compounds can also actallosterically and so they can also bind to the three proteins atpositions other than, and even remote from, the binding sites for MUC1(on HSP70 and HSP90) and on HSP70 or HSP90 (for MUC1). As usedthroughout the present application, the term “antibody” refers to awhole antibody (e.g., IgM, IgG, IgA, IgD, or IgE) molecule that isgenerated by any one of a variety of methods that are known in the art.The antibody can be made in or derived from any of a variety of species,e.g., humans, non-human primates (e.g., monkeys, baboons, orchimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, and mice.

The antibody can be a purified or a recombinant antibody. Also usefulfor the invention are antibody fragments and chimeric antibodies andhumanized antibodies made from non-human (e.g., mouse, rat, gerbil, orhamster) antibodies. As used herein, the term “antibody fragment” refersto an antigen-binding fragment, e.g., Fab, F(ab′)₂, Fv, and single chainFv (scFv) fragments. An scFv fragment is a single polypeptide chain thatincludes both the heavy and light chain variable regions of the antibodyfrom which the scFv is derived. In addition, diabodies (Poljak (1994)Structure 2(12):1121-1123; Hudson et al. (1999) J. Immunol. Methods23(1-2):177-189, the disclosures of both of which are incorporatedherein by reference in their entirety) and intrabodies (Huston et al.(2001) Hum. Antibodies 10(3-4):127-142; Wheeler et al. (2003) Mol. Ther.8(3):355-366; Stocks (2004) Drug Discov. Today 9(22): 960-966, thedisclosures of all of which are incorporated herein by reference intheir entirety) can be used in the methods of the invention.

Antibody fragments that contain the binding domain of the molecule canbe generated by known techniques. For example: F(ab′)₂ fragments can beproduced by pepsin digestion of antibody molecules; and Fab fragmentscan be generated by reducing the disulfide bridges of F(ab′)₂ fragmentsor by treating antibody molecules with papain and a reducing agent. See,e.g., National Institutes of Health, 1 Current Protocols In Immunology,Coligan et al., ed. 2.8, 2.10 (Wiley Interscience, 1991) the disclosureof which is incorporated herein by reference in their entirety. scFvfragments can be produced, for example, as described in U.S. Pat. No.4,642,334, the disclosure of which is incorporated herein by referencein its entirety.

Chimeric and humanized monoclonal antibodies can be produced byrecombinant DNA techniques known in the art, for example, using methodsdescribed in Robinson et al., International Patent PublicationPCT/US86/02269; Akira et al., European Patent Application 184,187;Taniguchi, European Patent Application 171,496; Morrison et al.,European Patent Application 173,494; Neuberger et al., PCT ApplicationWO 86/01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent Application 125,023; Better et al. (1988) Science 240,1041-43; Liu et al. (1987) J. Immunol. 139, 3521-26; Sun et al. (1987)PNAS 84, 214-18; Nishimura et al. (1987) Canc. Res. 47, 999-1005; Woodet al. (1985) Nature 314, 446-49; Shaw et al. (1988) J. Natl. CancerInst. 80, 1553-59; Morrison, (1985) Science 229, 1202-07; Oi et al.(1986) BioTechniques 4, 214; Winter, U.S. Pat. No. 5,225,539; Jones etal. (1986) Nature 321, 552-25; Veroeyan et al. (1988) Science 239, 1534;and Beidler et al. (1988) J. Immunol. 141, 4053-60. The disclosures ofall these articles and patent documents are incorporated herein byreference in their entirety.

Cells to which the method of the invention can be applied includegenerally any cell that expresses MUC1. Such cells include normal cells,such as any normal epithelial cell, or a cancer cell, the proliferationof which it is desired to inhibit. An appropriate cancer cell can be abreast cancer, lung cancer, colon cancer, pancreatic cancer, renalcancer, stomach cancer, liver cancer, bone cancer, hematological cancer(e.g., leukemia or lymphoma), neural tissue cancer, melanoma, ovariancancer, testicular cancer, prostate cancer, cervical cancer, vaginalcancer, or bladder cancer cell. In addition, the methods of theinvention can be applied to a wide range of species, e.g., humans,non-human primates (e.g., monkeys, baboons, or chimpanzees), horses,cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils,hamsters, rats, and mice.

The methods can be performed in vitro, in vivo, or ex vivo. In vitroapplication of appropriate compounds can be useful, for example, inbasic scientific studies of tumor cell biology, e.g., studies on themechanism of action of MUC1 and/or the MUC1-binders in promoting tumorcell growth, including survival. In addition, the compounds that areinhibitory can be used as “positive controls” in methods to identifyadditional compounds with inhibitory activity (see above). In such invitro methods, cells expressing MUC1 and one or more of theMUC1-binders, can be incubated for various times with the inhibitorycompound(s) at a variety of concentrations. Other incubation conditionsknown to those in art (e.g., temperature, or cell concentration) canalso be varied. Inhibition of binding can be tested by methods such asthose disclosed herein.

The methods of the invention will preferably be in vivo or ex vivo.

Compounds that inhibit binding between MUC1 and a MUC1-binder aregenerally useful as cancer cell (e.g., breast cancer cell)survival-inhibiting and/or cell cycle-arresting therapeutics orprophylactics. They can be administered to mammalian subjects (e.g.,human breast cancer patients) alone or in conjunction with other drugsand/or radiotherapy. The compounds can also be administered to subjectsthat are genetically and/or due to, for example, physiological and/orenvironmental factors, susceptible to cancer, e.g., subjects with afamily history of cancer (e.g., breast cancer), subjects with chronicinflammation or subject to chronic stress, or subjects that are exposedto natural or non-natural environmental carcinogenic conditions (e.g.,excessive exposure to sunlight, industrial carcinogens, or tobaccosmoke). As used herein, a compound that is “therapeutic” is a compoundthat causes a complete abolishment of the symptoms of a disease or adecrease in the severity of the symptoms of the disease. “Prevention”means that symptoms of the disease (e.g., cancer) are essentiallyabsent. As used herein, “prophylaxis” means complete prevention of thesymptoms of a disease, a delay in onset of the symptoms of a disease, ora lessening in the severity of subsequently developed disease symptoms.

When the methods are applied to subjects with cancer, prior toadministration of a compound, the cancer can optionally be tested forMUC1 expression (MUC1 protein or MUC1 mRNA expression) by methods knownin the art. In this way, subjects can be identified as having aMUC1-expressing cancer. Such methods can be performed in vitro on cancercells obtained from a subject. Alternatively, in vivo imaging techniquesusing, for example, radiolabeled antibodies specific for MUC1 can beperformed. In addition, body fluids (e.g., blood or urine) from subjectswith cancer can be tested for elevated levels of MUC1 protein or MUC1protein fragments.

In Vivo Approaches

In one in vivo approach, a compound that inhibits binding of MUC1 to aMUC1-binder is administered to a subject. Generally, the compounds ofthe invention will be suspended in a pharmaceutically-acceptable carrier(e.g., physiological saline) and administered orally or injectedintravenously, subcutaneously, intramuscularly, intrathecally,intraperitoneally, intrarectally, intravaginally, intranasally,intragastrically, intratracheally, or intrapulmonarily. They can also bedelivered directly to tumor cells, e.g., to a tumor or a tumor bedfollowing surgical excision of the tumor, in order to kill any remainingtumor cells. The dosage required depends on the choice of the route ofadministration; the nature of the formulation; the nature of thepatient's illness; the subject's size, weight, surface area, age, andsex; other drugs being administered; and the judgment of the attendingphysician. Suitable dosages are in the range of 0.0001 mg/kg-100 mg/kg.Wide variations in the needed dosage are to be expected in view of thevariety of compounds available and the differing efficiencies of variousroutes of administration. For example, oral administration would beexpected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2-, 3-, 4-,6-, 8-, 10-, 20-, 50-, 100-, 150-, or more fold). Encapsulation of thepolypeptide in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

Alternatively, where an inhibitory compound is a polypeptide, apolynucleotide containing a nucleic acid sequence encoding thepolypeptide can be delivered to appropriate cells in a mammal.Expression of the coding sequence can be directed to any cell in thebody of the subject. However, expression will preferably be directed tocells in the vicinity of the tumor cells whose proliferation it isdesired to inhibit. Expression of the coding sequence can be directed tothe tumor cells themselves. This can be achieved by, for example, theuse of polymeric, biodegradable microparticle or microcapsule deliverydevices known in the art.

Another way to achieve uptake of the nucleic acid is using liposomes,prepared by standard methods. The vectors can be incorporated alone intothese delivery vehicles or co-incorporated with tissue-specific ortumor-specific antibodies. Alternatively, one can prepare a molecularconjugate composed of a plasmid or other vector attached topoly-L-lysine by electrostatic or covalent forces. Poly-L-lysine bindsto a ligand that can bind to a receptor on target cells (Cristiano etal. (1995) J. Mol. Med. 73:479, the disclosure of which is incorporatedherein by reference in its entirety). Alternatively, tissue specifictargeting can be achieved by the use of tissue-specific transcriptionalregulatory elements (TRE) which are known in the art. Delivery of “nakedDNA” (i.e., without a delivery vehicle) to an intramuscular,intradermal, or subcutaneous site is another means to achieve in vivoexpression.

In the relevant polynucleotides (e.g., expression vectors), the nucleicacid sequence encoding the polypeptide of interest with an initiatormethionine and optionally a targeting sequence is operatively linked toa promoter or enhancer-promoter combination. Short amino acid sequencescan act as signals to direct proteins to specific intracellularcompartments. Such signal sequences are described in detail in U.S. Pat.No. 5,827,516, the disclosure of which is incorporated herein byreference in its entirety.

Enhancers provide expression specificity in terms of time, location, andlevel. Unlike a promoter, an enhancer can function when located atvariable distances from the transcription initiation site, provided apromoter is present. An enhancer can also be located downstream of thetranscription initiation site. To bring a coding sequence under thecontrol of a promoter, it is necessary to position the translationinitiation site of the translational reading frame of the peptide orpolypeptide between one and about fifty nucleotides downstream (3′) ofthe promoter. Promoters of interest include but are not limited to thecytomegalovirus hCMV immediate early gene, the early or late promotersof SV40 adenovirus, the lac system, the trp system, the TAC system, theTRC system, the major operator and promoter regions of phage A, thecontrol regions of fd coat protein, the promoter for 3-phosphoglyceratekinase, the promoters of acid phosphatase, and the promoters of theyeast α-mating factors, the adenoviral E1b minimal promoter, or thethymidine kinase minimal promoter. The DF3 enhancer can be particularlyuseful for expression of an inhibitory compound in cells that naturallyexpress MUC1, for example, normal epithelial cells or malignantepithelial cells (carcinoma cells), e.g., breast cancer cells (see U.S.Pat. Nos. 5,565,334 and 5,874,415, the disclosures of which areincorporated herein by reference in their entirety). The coding sequenceof the expression vector is operatively linked to a transcriptionterminating region.

Suitable expression vectors include plasmids and viral vectors such asherpes viruses, retroviruses, vaccinia viruses, attenuated vacciniaviruses, canary pox viruses, adenoviruses and adeno-associated viruses,among others.

Polynucleotides can be administered in a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are biologicallycompatible vehicles that are suitable for administration to a human,e.g., physiological saline or liposomes. A therapeutically effectiveamount is an amount of the polynucleotide that is capable of producing amedically desirable result (e.g., decreased proliferation of cancercells) in a treated animal. As is well known in the medical arts, thedosage for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Dosages will vary, but apreferred dosage for administration of polynucleotide is fromapproximately 10⁶ to approximately 10¹² copies of the polynucleotidemolecule. This dose can be repeatedly administered, as needed. Routes ofadministration can be any of those listed above.

Ex Vivo Approaches

An ex vivo strategy can involve transfecting or transducing cellsobtained from the subject with a polynucleotide encoding a polypeptidethat inhibit binding of MUC1 to a MUC1-binder. The transfected ortransduced cells are then returned to the subject. The cells can be anyof a wide range of types including, without limitation, hemopoieticcells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells,T cells, or B cells), fibroblasts, epithelial cells, endothelial cells,keratinocytes, or muscle cells. Such cells act as a source of theinhibitory polypeptide for as long as they survive in the subject.Alternatively, tumor cells, preferably obtained from the subject butpotentially from an individual other than the subject, can betransfected or transformed by a vector encoding the inhibitorypolypeptide. The tumor cells, preferably treated with an agent (e.g.,ionizing irradiation) that ablates their proliferative capacity, arethen introduced into the patient, where they secrete the polypeptide.

The ex vivo methods include the steps of harvesting cells from asubject, culturing the cells, transducing them with an expressionvector, and maintaining the cells under conditions suitable forexpression of the polypeptide that inhibits inhibit binding of MUC1 to aMUC1-binder or phosphorylation of MUC1 by a MUC1-binder. These methodsare known in the art of molecular biology. The transduction step isaccomplished by any standard means used for ex vivo gene therapy,including calcium phosphate, lipofection, electroporation, viralinfection, and biolistic gene transfer. Alternatively, liposomes orpolymeric microparticles can be used. Cells that have been successfullytransduced can be selected, for example, for expression of the codingsequence or of a drug resistance gene. The cells may then be lethallyirradiated (if desired) and injected or implanted into the patient.

In any of the above methods of inhibiting the interaction between MUC1and a MUC1-binder and of inhibiting expression of MUC1, one or moreagents (e.g., two, three, four, five, six, seven, eight, nine, ten, 11,12, 15, 18, 20, 25, 30, 40, 50, 60, 70, 80, 100, or more) including, forexample, inhibitory compounds, antisense oligonucleotides, siRNA, drugs,aptamers, or other small molecules (or vectors encoding them), can beused.

The above in vivo and ex vivo methods of inhibiting interactions betweenMUC1 and MUC1-binders and inhibiting expression of MUC1 can be used inconjunction with any of a variety of other cancertherapeutic/prophylactic regimens (e.g., chemotherapeutic,radiotherapeutic, biotherapeutic/prophylactic, andimmunotherapeutic/prophylactic regimens). Of particular interest areregimens involving genotoxic (DNA-damaging) agents. Such agents includevarious forms of ionizing and non-ionizing radiation and a variety ofchemotherapeutic compounds.

Non-ionizing radiation includes, for example, ultra-violet (UV)radiation, infra-red (IR) radiation, microwaves, and electronicemissions. The radiation employed in the methods of the invention ispreferably ionizing radiation. As used herein, “ionizing radiation”means radiation composed of particles or photons that have sufficientenergy or can produce sufficient energy by atomic nuclear interactionsto produce ionization (gain or loss of electrons) of an atom. Ionizingradiation thus includes, without limitation, α-radiation, β-radiation,γ-radiation, or x-radiation. A preferred radiation is x-radiation.

Ionizing radiation causes DNA damage and cell killing generally inproportion to the dose administered. It has been indicated that themultiple biological effects induced by ionizing radiation are due eitherto the direct interaction of the radiation with DNA or to the formationof free radical species which lead to damage of DNA. These effectsinclude gene mutations, malignant transformation, and cell killing.

External and internal means for delivering ionizing radiation to atarget tissue or cell are known in the art. External sources include βor γ sources or linear accelerators and the like. Alternatively,ionizing radiation may be delivered, for example, by administering aradiolabeled antibody that is capable of binding to a molecule expressedon the surface of a carcinoma (e.g., MUC1 or Her2/neu) to a subject, orby implantation of radiation-emitting pellets in or near the tumor(brachytherapy).

The amount of radiation (e.g., ionizing radiation) needed to kill agiven cell generally depends upon the nature of the cell. As usedherein, an “effective dose” of radiation means a dose of radiation thatproduces cell damage or death when given in conjunction with anadenoviral vector of the invention. Means of determining an effectivedose are known in the art. Dosage ranges for x-radiation range fromdaily doses of 50 to 200 roentgens for prolonged periods of time (e.g.,6-8 weeks or even longer) to single doses of 2,000 to 6,000 roentgens.Dosages for administered radioisotopes vary widely, and depend on thehalf-life of the isotope, the strength and type of radiation emitted,and the degree of uptake by the target cells.

As used herein, “chemotherapeutic agents” are chemical compounds thatare useful for inhibiting the growth, proliferation, or division ofcancer cells, e.g., agents that enter cells and damage DNA. Thus, theycan be compounds which, for example, directly cross-link DNA (e.g.,cisplatin (CDDP) and other alkylating agents), intercalate into DNA, orinterfere with DNA replication, mitosis, or chromosomal segregation,e.g., adriamycin (also known as doxorubicin), VP-16 (also known asetoposide), podophyllotoxin, and the like. These compounds are widelyused in the treatment of carcinomas. Chemotherapeutic agents useful inthe methods of the invention include, without limitation, cisplatin,carboplatin, procarbazine, mechlorethamine, cyclophosphamide,camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,mitomycin, etoposide, podophyllotoxin, tamoxifen, taxol, transplatinum,5-fluorouracil, vincristin, vinblastin, methotrexate, or any analog orderivative of these that is effective in damaging DNA.

Routes of administration are the same as those disclosed herein forinteraction-inhibiting and expression-inhibiting compounds. Doses andfrequency of administration vary widely according to all the variableslisted above for administration of interaction-inhibiting andexpression-inhibiting compounds. For example, adriamycin can beadministered by bolus intravenous injection at doses in the range of25-75 mg/kg and etoposide can be administered intravenously or orally atdoses in the range of 35-100 mg/kg. Methods of determining optimalparameters of administration are well known in the art.

Combination treatments can include administration of one or more (e.g.,one, two, three, four, five, six, seven, eight, nine or ten)interaction-inhibiting and/or expression-inhibiting compounds of theinvention, and one or more (e.g., one, two, three, four, five, six,seven, eight, nine or ten) radiation modalities, and/or one or more(e.g., one, two, three, four, five, six, seven, eight, nine or ten)chemotherapeutic agents. The interaction-inhibiting andexpression-inhibiting compounds, radiation treatments andchemotherapeutic agents can be given in any order and frequency. Theycan be given simultaneously or sequentially. Treatment with any one ofthe modalities (interaction-inhibiting and expression-inhibitingcompounds, radiation, or chemotherapeutic agents) can involve single ormultiple (e.g., two, three, four, five, six, eight, nine, ten, 12, 15,20, 30, 40, 50, 60, 80, 100, 200, 300, 500, or more) administrationsseparated by any time period found to be optimal in terms of therapeuticbenefit. Multiple administrations can be separated by one to 23 hours, aday, two, three days, four days, five days, six days, seven days, eight,ten days, twelve days, two weeks, three weeks four weeks, a month, twomonths, three months, four months, five months, six months, sevenmonths, eight months, nine months, ten months, eleven months, a year,one and one half of a year, two years, three years, five years, or tenyears. Administrations can be continued for as long as the subject isneed of the treatment, e.g., any of the of the above time intervals, andcan be for the life of the subject. Administrations can be, for example,once a week for the life-time of the subject. When administrations ofany or all of the modalities are multiple, the course of any one can besimultaneous with, overlapping with, or consequent to the course(s) ofthe other(s).

The invention is illustrated, and not limited, by the followingexamples.

EXAMPLES Example 1 Materials and Methods

Cell culture. Human HCT116/vector, HCT116/MUC1, HCT116/MUC1 (Y46F) (Renet al. (2004) Cancer Cell 5:163-175) and HCT116/MUC1-CD (Huang et al.(2003) Cancer Biol. Ther. 2:702-706) cells were cultured in Dulbecco'sModified Eagle Medium/F12 with 10% heat-inactivated fetal bovine serum(HI-FBS), 100 units/ml penicillin, 100 mg/ml streptomycin, and 2 mML-glutamine. Human HeLa cervical carcinoma, 293 embryonic kidney andMCF-7 breast cancer cells were grown in Dulbecco's Modified Eagle Mediumcontaining 10% HI-FBS, antibiotics and L-glutamine. Human ZR-75-1 breastcancer cells were cultured in RPMI1640 medium containing 10% HI-FBS,antibiotics and L-glutamine. Cells were grown to 60% confluence and thenmaintained overnight in medium with 0.1% serum before treatment withheregulin (HRG; 20 ng/ml; Calbiochem-Novabiochem, LaJolla, Calif.).Cells were also treated with PP2 (10 TM; Calbiochem-Novabiochem) orgeldanamycin (GA; 1 TM; Calbiochem-Novabiochem).

Tandem affinity purification of MUC1 protein complexes. The procedurefor purification and analysis of intracellular protein complexes hasbeen described (Ogawa et al. (2002) Science 296:1132-6; Shi et al.(2003) Nature 422:735-8). In brief, MUC1-C was cloned downstream to HAand Flag tags in the retroviral pOZ-N vector (Nakatani and Ogryzko(2003) Methods Enzymol. 370:430-444), which expresses the IL-2 receptor(FIG. 1A). HeLa cells were transduced with retroviruses expressing MUC1or the empty control vector and selected with IL-2-coupled magneticbeads. Intracellular protein complexes were purified by affinitychromatography using anti-Flag conjugated to beads. The adsorbedproteins were separated by centrifugation in 10-40% glycerol gradients.The gradient fractions reactive by immunoblotting with anti-Flag weresubjected to electrophoresis in 4-20% polyacrylamide/SDS gels andstained with Coomassie blue. Proteins bands were excised and analyzed byMALDI-TOF-MS.

Immunoprecipitation and immunoblotting. Equal amounts of protein fromcell lysates were incubated with anti-MUC1-N (antibody DF3) (Kufe et al.(1984) Hybridoma 3:223-232), anti-Flag (Sigma-Aldrich, St. Louis, Mo.),anti-c-Src (Upstate Biotechnology, Lake Placid, N.Y.), anti-HSP90 (BDBiosciences PharMingen, San Diego, Calif.) or normal mouse IgG (SantaCruz Biotechnology, Santa Cruz, Calif.) for 2 hours at 4° C. Immunecomplexes were prepared as described (Li et al. (1998) Mol. Cell. Biol.18:7216-7224) and subjected to immunoblot analysis with anti-HSP70,anti-HSP90, anti-MUC1-N, anti-MUC1-C (Ab5; Neomarkers, Fremont, Calif.),anti-phospho-c-Src(Tyr416) (Cell Signaling Technology, Beverly, Mass.)or anti-c-Src. Lysates not subjected to immunoprecipitation wereimmunoblotted with anti-MUC1-C, anti-HSP60 (Stressgen Biotechnologies,Victoria, BC, Canada), anti-PDGFR (Santa Cruz Biotechnology), anti-BAP31(Abcam, Cambridge, Mass.), anti-PCNA (Calbiochem-Novabiochem), anti-IkBa(Santa Cruz Biotechnology), anti-calreticulin (StressgenBiotechnologies), anti-Tom20 (BD Biosciences), anti-Tim23 (BDBiosciences), anti-Tim44 (BD Biosciences) or anti-θ-actin(Sigma-Aldrich). Reactivity was detected with horseradishperoxidase-conjugated secondary antibodies and chemiluminescence(PerkinElmer Life Sciences, Boston, Mass.). Intensity of the signals wasdetermined by densitometric scanning Statistical significance wasdetermined by the Student's t-test.

Cell transfections. 293 cells were transiently transfected withpIRES-puro2-MUC1, pIRES-puro2-MUC1 (Y46F), or pCMV-c-Src (Li et al.(2001) J. Biol. Chem. 276:6061-6064) in the presence of LipofectAMINE3(Invitrogen Life Technologies, Carlsbad, Calif.).

Binding Studies. Sequences encoding MUC1-CD, MUC1-CD (1-45), or MUC1-CD(46-72) were amplified by PCR and cloned into the pGEX-4T vector(Amersham Biosciences, Piscataway, N.J.). Purified GST-MUC1-CD fusionproteins bound to glutathione beads were incubated with purifiedrecombinant human HSP70 or HSP90 (Stressgen Biotechnologies) for 1 hourat 4° C. In other experiments, the purified GST fusion proteins bound toglutathione beads were incubated with or without 50 ng c-Src (UpstateBiotechnology) in the presence of 200 mM ATP for 20 minutes at 30° C.HSP90 was then added for 1 hour at 4° C. Precipitated proteins weresubjected to immunoblot analysis with anti-HSP70 or anti-HSP90.

Subcellular fractionation. Cell membranes and mitochondria were purifiedas described (Kharbanda et al. (1996) Cancer Res. 56:3617-3621; Ren etal. (2004) Cancer Cell 5:163-175). Cytosolic fractions were purified asdescribed (Datta et al. (2000) J. Biol. Chem. 275:31733-31738; Kharbandaet al. (1996) Cancer Res. 56:3617-3621). The mitochondria (200 mg) weresuspended in 40 ml SM buffer (10 mM MOPS-KOH, pH 7.2, 250 mM sucrose)and then divided into two equal aliquots (Ryan et al. (2001) MethodsCell Biol. 65:189-215). SM buffer (180 ml) was added to one aliquot, andhypotonic buffer (10 mM MOPS-KOH, pH 7.2; 180 ml) was added to the otheraliquot. The samples were incubated for 15 minutes on ice and then leftuntreated or digested with 60 mg/ml trypsin (Sigma-Aldrich) for 15minutes at 4° C. In other experiments, purified mitochondria insuspension buffer (5 mM HEPES, pH 7.4, 210 mM mannitol, 70 mM sucrose,100 mM KCl, and 1 mM EGTA) were treated with 0.5% or 1% digitonin(Sigma-Aldrich) for 15 minutes at 4° C. Alternatively, the mitochondriawere incubated with 0.5% digitonin for 1 minute at 4° C., diluted withsuspension buffer, and then treated with 60 mg/ml trypsin for 15 minutesat 4° C.

Example 2 Tandem Affinity Purification of MUC1 Protein Complexes

To identify proteins that associate with MUC1, MUC1-C was cloneddownstream of Flag and HA tags in the retroviral pOZ-N vector (FIG. 1A).Lysates from HeLa cells stably transduced with the MUC1 retrovirus orthe empty retrovirus were subjected to affinity chromatography withanti-Flag conjugated to beads. The adsorbed protein complexes wereseparated by glycerol gradient centrifugation. Analysis of Flag-positivegradient fractions by SDS-PAGE and Coomassie blue staining showedmultiple proteins that associated with MUC1 (FIG. 1B). Identification oftwo MUC1-associated proteins with molecular masses of ˜70 and 90 kDa wasdetermined by MALDI-TOF-MS. Mass fingerprinting and sequencing ofselected peptides demonstrated identity of the smaller protein withHSP70 (FIG. 1C). The other protein was identified as HSP90 (FIG. 1D). Bycontrast, HSP70 and HSP90 were undetectable following the same analysisof proteins immunoprecipitated with anti-Flag from cells expressing theempty retroviral vector. These findings indicated that MUC1 formsintracellular complexes with the HSP70 and HSP90 chaperones.

Example 3 MUC1 Binds to HSP70 and HSP90

To confirm the association of MUC1 with HSP70 and HSP90, lysates fromHCT116 cells that stably express MUC1 were immunoprecipitated withanti-MUC1-N or a control IgG. Immunoblot analysis of the precipitateswith anti-HSP70 or anti-HSP90 confirmed that MUC1 forms complexes withboth chaperones (FIG. 2A). Intensity of the HSP70 and HSP90 signals, asdetermined by scanning densitometry, indicated that MUC1 associated withapproximately 0.6% and 0.4% of the total HSP70 and HSP90 pools,respectively. To determine if the MUC1 cytoplasmic domain (MUC1-CD)confers the association with HSP70 and/or HSP90, lysates from HCT116cells stably expressing Flag-MUC1-CD were immunoprecipitated withanti-Flag. Immunoblot analysis of the precipitates demonstrated thatMUC1-CD associates with both HSP70 and HSP90 (FIG. 2B). To investigatewhether MUC1 interacts directly with HSP70 or HSP90, GST or GST-MUC1-CDwas incubated with purified recombinant HSP proteins (FIG. 2C).Immunoblot analysis of the adsorbates showed that MUC1-CD binds toHSP70, but not HSP90 (FIG. 2D). When deletion mutants of MUC1-CD wereused in similar reactions, binding to HSP70 was detected with MUC1-CD(46-72), but not MUC1-CD (1-45) (FIG. 2D), indicating that theC-terminal region of MUC1-CD confers the interaction.

MUC1 is phosphorylated on Y-46 by c-Src and the pYEKV motif functions asa binding site for the c-Src SH2 domain (Li et al. (2001) J. Biol. Chem.276:6061-6064) (FIG. 2C). To determine if c-Src regulates binding ofMUC1-CD to HSP70 or HSP90, MUC1 was incubated with c-Src and ATP, andthen the recombinant HSPs were added for binding reactions at 4° C.c-Src had little, if any, effect on binding of MUC1-CD and HSP70.However, c-Src induced binding of MUC1-CD to HSP90 (FIG. 2E). As acontrol, there was no detectable binding of HSP90 to the GST-MUC1-CD(1-45) deletion mutant (FIG. 2E). Moreover, there was no detectablebinding of HSP90 to GST-MUC1-CD (46-72) which is phosphorylated byc-Src, suggesting that the sequences surrounding the MUC1 Y-46 site areof importance for the interaction with HSP90 (FIG. 2E). Of note, bindingof MUC1-CD to HSP90 was performed in the absence of HSP70, indicatingthat HSP70 was dispensable for the MUC1-HSP90 interaction in vitro.These findings indicate that MUC1-CD binds directly to HSP70 and thatbinding of MUC1-CD to HSP90 in vitro is mediated by a c-Src-dependentmechanism.

Example 4 Activation of c-Src and Phosphorylation of MUC1 on Y-46Confers Binding to HSP90 In Vivo

When MUC1 was expressed in 293 cells in the absence or presence ofc-Src, there was little effect of c-Src on binding of MUC1 to HSP70(FIG. 3A). Moreover, expression of MUC1 with a Y46F mutation had noapparent effect on binding of MUC1 to HSP70 in the absence or presenceof c-Src (FIG. 3A). By contrast, c-Src stimulated binding of MUC1 toHSP90, and the Y46F mutation attenuated this response (FIG. 3A). Thesefindings were confirmed in repeated experiments (FIG. 3B). As determinedby autophosphorylation, HRG activated c-Src in HCT116/MUC1 cells (FIG.3C). In addition, treatment of the HCT116/MUC1 cells with PP2, a c-Srcinhibitor, blocked HRG-induced c-Src activation (FIG. 3C). Consequently,it was examined if HRG regulates binding of MUC1 to HSP90. Indeed, HRGstimulation of the HCT116/MUC1 cells was associated with an increase inbinding of MUC1 and HSP90 (FIG. 3D). Consistent with involvement ofc-Src, PP2 attenuated this response (FIG. 3D). As a control, inhibitionof HSP90 with geldanamycin (GA), an inhibitor of HSP90 function(Whitesell et al. (1994) Proc. Natl. Acad. Sci. USA 91:8324-8), alsoattenuated the HRG-induced interaction between MUC1 and HSP90 (FIG. 3D).Similar results were obtained in repeated experiments (FIG. 3E). Toconfirm these results and further assess involvement of the Y-46 site,anti-HSP90 precipitates from control and HRG-treated HCT116/MUC1 cellswere immunoblotted for MUC1. The results show that HRG increased theformation of MUC1-HSP90 complexes (FIG. 3F). As a control, MUC1 was notdetectable in anti-HSP90 immunoprecipitates from HCT116/vector cells(FIG. 3F). Previous work has shown that HCT116 cells stably expresssimilar levels of wild-type MUC1 and MUC1 (Y46F) (Ren et al. (2004)Cancer Cell 5:163-175). Notably, the interaction between MUC1 and HSP90was attenuated by the MUC1 Y46F mutation in control and HRG-treatedcells (FIG. 3F). HRG-induced binding of MUC1 to HSP90, and attenuationof this response with MUC1 (Y46F) was confirmed in repeated experiments(FIG. 3G). These findings indicate that activation of c-Src by HRG, andthereby phosphorylation of MUC1 on Y-46, stimulates the interactionbetween MUC1 and HSP90.

Example 5 HRG Induces Binding of Endogenous MUC1 and HSP90

To determine if HRG also stimulates binding of endogenous MUC1 andHSP90, lysates from human MCF-7 breast cancer cells wereimmunoprecipitated with anti-MUC1-N. Immunoblot analysis of theprecipitates demonstrated that endogenous MUC1 associates with HSP70 andthat HRG has little if any effect on this interaction (FIGS. 4A and 4B).Moreover, as found in HCT116/MUC1 cells, stimulation of MCF-7 cells withHRG was associated with increased binding of MUC1 and HSP90 (FIGS. 4Aand 4B). In the reciprocal experiment, immunoblot analysis of anti-HSP90precipitates with anti-MUC1-C confirmed that HRG stimulates binding ofMUC1-C and HSP90 (FIGS. 4C and 4D). Studies with human ZR-75-1 breastcancer cells that express endogenous MUC1 also demonstrated that HRG hasno apparent effect on the constitutive interaction between MUC1 andHSP70 (FIGS. 4E and 4F). However, as found in MCF-7 cells, HRGstimulation was associated with increased binding of MUC1 and HSP90(FIGS. 4E and 4F). These results were confirmed when anti-HSP90precipitates from control and HRG-stimulated ZR-75-1 cells wereimmunoblotted with anti-MUC1-C (FIGS. 4G and 4H). These findingsindicate that HRG stimulates the interaction between endogenous MUC1 andHSP90, but not HSP70.

Example 6 HRG Stimulates Binding of MUC1 to HSP90 at the Cell Membraneand in the Cytosol

MUC1 is a cell membrane-associated protein that also accumulates in thecytosol of transformed cells (Croce et al. (2003) J. Histochem.Cytochem. 51:781-8; Kufe et al. (1984) Hybridoma 3:223-232; Perey et al.(1992) Cancer Res. 52:2563-3568; Rahn et al. (2001) Cancer 91:1973-82).To assess the subcellular location where MUC1 interacts with HSP90,whole cell lysates and purified cell membranes from control andHRG-stimulated HCT116/MUC1 cells were immunoprecipitated withanti-MUC1-N. Immunoblot analysis of the precipitates with anti-HSP90showed that, as found for whole cell lysates, HRG stimulates binding ofMUC1 and HSP90 at the cell membrane (FIGS. 5A and 5B). HRG stimulationwas also associated with an increase in cytosolic MUC1-C levels (FIGS.5C and 5D). The purity of the cytosolic fraction was confirmed byimmunoblotting with antibodies against the cell membrane-associatedplatelet-derived growth factor receptor (PDGFR), endoplasmic reticulum(ER)-associated BAP31 (Ng et al. (1997) J. Cell. Biol. 139:327-38), andthe mitochondria-associated Tom20 proteins (FIGS. 5C and 5D). In concertwith these results, it was also found that HRG increases binding ofcytosolic MUC1-C and HSP90 (FIGS. 5E and 5F). To confirm that HRGstimulates binding of cytosolic MUC1 to HSP90, HCT116 cells were studiedthat stably express Flag-MUC1-CD, which is devoid of the transmembranedomain and is expressed in the cytosol (Huang et al. (2003) Cancer Biol.Ther. 2:702-706). HRG stimulation of the HCT116/MUC1-CD cells had littleeffect on binding of MUC1-CD to HSP70 (FIGS. 5G and 5H). However, likefull-length MUC1, HRG stimulated binding of MUC1-CD to HSP90 (FIGS. 5Gand 5H), confirming that cytosolic MUC1 formed a complex with HSP90.These findings indicate that HRG stimulates binding of MUC1 to HSP90 atthe cell membrane and in the cytosol.

Example 7 c-Src and HSP90 Regulate Delivery of MUC1 to Mitochondria

HRG stimulation is associated with targeting of MUC1-C to mitochondria(Ren et al. (2004) Cancer Cell 5:163-175). To assess the involvement ofc-Src in mitochondrial targeting of MUC1-C, mitochondria were purifiedfrom HCT116/MUC1 cells stimulated with HRG in the absence and presenceof PP2. The results confirm that HRG induces localization of MUC1-C, andnot MUC1-N, to mitochondria and show that this response is attenuated byPP2 (FIG. 6A). Equal loading of the mitochondrial lysates was confirmedby immunoblotting for the mitochondrial HSP60 protein (FIG. 6A).Densitometric scanning of the constitutive mitochondrial MUC1-C signalindicated that 0.5 to 1% of total cellular MUC1-C localized to themitochondria. Treatment with GA also blocked HRG-induced targeting ofMUC1 to mitochondria, consistent with the involvement of HSP90 in thisresponse (FIG. 6B). In addition purity of the mitochondria was confirmedby immunoblotting with antibodies against the nuclear PCNA, cytosolicIκBα, and ER-associated calreticulin proteins (FIG. 6B). As anadditional control, there was no detectable ErbB2 in the purifiedmitochondrial fraction. To assess stability of MUC1 that constitutivelyresides in mitochondria, HCT116/MUC1 cells were treated with GA for 4,8, and 16 hours (FIG. 6C). Decreases in mitochondrial MUC1 levels weredetectable with GA exposures of 8 and 16 hours (FIG. 6C). Densitometricscanning of the signals indicated that GA decreases mitochondrial MUC1with a half-life of approximately 8 hours. As a control, similarexposures to GA had little effect on total intracellular MUC1 levels(FIG. 6C). These findings indicate that MUC1 is targeted to mitochondriaby a mechanism dependent on c-Src and HSP90.

Example 8 MUC1 is Integrated into the Mitochondrial Outer Membrane

Complexes of HSP70 and HSP90 function in the transport of proteins, suchas MUC1, without N-terminal mitochondrial localization sequences to themitochondrial surface (Young et al. (2003) Cell 112:41-50). Previouswork has demonstrated that mitochondrial MUC1 is an integral membraneprotein that is resistant to trypsin digestion (Ren et al. (2004) CancerCell 5:163-175). To further define the localization of MUC1, purifiedmitochondria were incubated in hypotonic buffer to induce swelling anddisruption of the outer membrane (FIG. 7A). As shown previously (Ren etal. (2004) Cancer Cell 5:163-175), treatment of mitochondria withtrypsin had no effect on MUC1 but decreased Tom20, a component of thetranslocase of the outer membrane (FIG. 7A). Of note, the N-terminus ofTom20 is anchored in the mitochondrial outer membrane and, as such, theTom20 C-terminal region is susceptible to protease digestion. Trypsindigestion had no apparent effect on Tim23, a component of themitochondrial inner membrane (FIG. 7A). After hypotonic disruption ofthe outer membrane, trypsin treatment had no effect on MUC1, but wasassociated with partial decreases in Tim23 levels and not the matrixHSP60 protein, consistent with access of the protease to themitochondrial inner membrane (FIG. 7A). In this regard, the region ofTim23 recognized by anti-Tim23 extends into the intermembrane space fromthe surface of the mitochondrial inner membrane (Moro et al. (1999) EMBOJ. 18:3667-75). To further assess the localization of MUC1, themitochondrial outer membrane was permeabilized with digitonin. Treatmentwith 0.5 or 1.0% digitonin alone had no effect on MUC1 or mitochondrialproteins associated with the outer (Tom20) or inner (Tim23 and Tim44)membranes (FIG. 7B). However, treatment of the mitochondria with bothdigitonin and trypsin resulted in complete digestion of MUC1, Tom20 andTim23 (FIG. 7C). By contrast, digitonin and trypsin had little effect onTim44, a protein associated with matrix face of the mitochondrial innermembrane (FIG. 7C). These findings collectively indicate that MUC1 isembedded in the mitochondrial outer membrane.

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1-13. (canceled)
 14. An in vivo method of inhibiting binding of MUC1 toHSP70 or HSP90 in a cancer cell that expresses MUC1, the methodcomprising: (a) identifying a subject as having a cancer that expressesMUC1 or is suspected to express MUC1; and (b) administering to thesubject a compound or, where the compound is a polypeptide, a nucleicacid comprising a nucleic acid sequence encoding the polypeptide, thenucleic acid sequence being operably linked to a transcriptionalregulatory element (TRE), wherein the compound inhibits binding of HSP70or HSP90 to the cytoplasmic domain of MUC1.
 15. The method of claim 14,wherein the compound is a peptide fragment of (a) MUC1, (b) HSP70, or(c) HSP90.
 16. The method of claim 14, wherein the compound is a peptidefragment of the cytoplasmic domain of MUC1.
 17. The method of claim 15,wherein the peptide fragment comprises all or part of amino acids 46-72of SEQ ID NO:1.
 18. The method of claim 15, wherein the peptidecomprises all or part of the substrate binding domain of HSP70 or HSP90.19. The method of claim 14, wherein the compound is an antibody, or anantibody fragment, that binds to the cytoplasmic domain of MUC1.
 20. Themethod of claim 14, wherein the compound is a small molecule.
 21. Themethod of claim 20, wherein the small molecule comprises a nucleic acidaptamer.
 22. The method of claim 20, wherein the small molecule consistsof a nucleic acid aptamer.
 23. The method of claim 14, wherein thesubject is a human subject.
 24. The method of claim 14, wherein thecancer cell is a breast cancer cell.
 25. The method of claim 14, whereinthe cancer cell is selected from the group consisting of a lung cancer,colon cancer, pancreatic cancer, renal cancer, stomach cancer, livercancer, bone cancer, hematological cancer, neural tissue cancer,melanoma, ovarian cancer, testicular cancer, prostate cancer, cervicalcancer, vaginal cancer, or bladder cancer cell.
 26. The method of claim21, wherein the TRE is a DF3 enhancer.
 27. A method of killing a cancercell, the method comprising before, after, or at the same time asperforming the method of claim 14, exposing the subject to one or moregenotoxic agents.
 28. The method of claim 27, wherein the one or moregenotoxic agents comprise one or more forms of ionizing radiation. 29.The method of claim 27, wherein the one or more genotoxic agentscomprise one or more chemotherapeutic agents.
 30. The method of claim29, wherein the one or more chemotherapeutic agents are selected fromthe group consisting of cisplatin, carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil,podophyllotoxin, tamoxifen, taxol, transplatinum, 5-fluorouracil,vincristin, vinblastin, methotrexate, and an analog of any of theaforementioned.